Notes of Molecular Biology of the Cell by Alberts

martyna p Theory

Molecular Biology of the Cell

Molecular Biology of the Cell by Alberts is a recommended textbook for the biology olympiad. Below you will find a great summary of the key chapters from the textbook.

Chapter 1 Introduction to Cells pp 1-38


Human Cells

  • 210+ cell types in body
  • total number of estimated cells in the body – 1013 (American Ten trillion/British Ten billion)

Flora

  • bacteria, fungi and archaea
  • found on all surfaces exposed to the environment
    • skin and eyes, in the mouth, nose, small intestine
  • most bacteria live in the large intestine
  • 500 to 1000 species of bacteria live in the human gut
  • total number of estimated flora ten times as many bacteria 1014 (American One hundred trillion/British One hundred billion)

Cell Sizes

  • frog or fish egg are the largest individual cells easily visible, approx 1+ mm diameter
  • human or sea urchin egg, approx 100 micron (µm) diameter
  • typical somatic cell, approx 20 micron diameter
  • plant cells are larger, approx 30 x 20 micron
  • bacteria are smaller, approx 2 x 1 micron

Divisions of Life

Prokaryotic

  • bacteria and archaea (single-celled microorganisms previously called archaebacteria)
    • no cell nucleus or any other organelles within their cells
    • organisms that can live in extreme habitats

Eukaryotic

  • plants
  • animals
  • fungi
  • protists

Unicellular and Multicellular

  • Unicellular
    • All prokaryotes and some eukaryotes
      • Yeast + budding, non-budding
      • Protozoa + classified by means of locomotion: flagellates, amoeboids, sporozoans, ciliates + often “feed” on bacteria
  • Multicellular
    • Eukaryotes
    • Plants and Animals
    • Allowed development of specialized cells
    • functions and tissues

Prokaryote

  • evolutionarily arose first (3.5 billion years ago) Evolution of Cells
  • bacteria are smaller, approx 2 x 1 micron (1×10-6 m)
  • not all bacteria are dangerous or disease causing

(MH – the adult human in addition bacteria to the skin surface and lining of the respiratory/digestive tract, also has intestines contains trillions of bacteria made up from hundreds of species and thousands of subspecies)

  • biochemically diverse
  • simple structure, classified by shape (rod-shaped, spherical or spiral-shaped)
  • some prokaryotic cells have also been shown to have a “cytoskeleton”, which is different from eukaryotic cells.
  • some bacteria are highly motile

Prokaryotes Cell Wall

  • Bacterial Shape – Bacterial shapes and cell-surface structures
  • Bacterial Membranes – A small section of the double membrane of an E. coli bacterium
    • Bacterial outer membranes – outer membrane contains porins
  • Bacterial cell walls – Bacterial cell walls
    • Gram-negative bacteria surrounded by a thin cell wall beneath the outer membrane
    • Gram-positive bacteria lack outer membranes and have thick cell walls

(MH – note that some unicellular eukaryotes can also have a cell wall)

  • Antibiotics – inhibit either bacterial protein synthesis or bacterial cell wall synthesis Antibiotic targets Gram-positive and Gram-negative bacteria
  • Bacterial Replication – DNA replication and cell division in a prokaryote

Prokaryote Mycoplasmas

  • smallest self-replicating organisms
  • smallest genomes (approx 500 to 1000 genes)
  • spherical to filamentous cells
  • no cell walls
  • surface parasites of the human respiratory and urogenital tracts
    • Mycoplasma pneumoniae infect the upper and lower respiratory tract
    • Mycoplasma genitalium a prevalent sexually transmitted infection
    • Mycoplasma hyorhinis found in patients with AIDS

Prokaryotic and Eukaryotic Cells

The following links describe the major differences between prokaryotic and eukaryotic cells, the way they divide and the way in which antibiotics have their action on prokaryotic cells.

Plant Cell

  • Plant cells are larger than mammalian cells approx 30 x 20 micron
  • Central Vacuole
    • tonoplast maintains cell’s turgor
    • storage (water, ions, and nutrients such as sucrose and amino acids, and waste products)
  • Plastids
    • organelles found in plants and algae
    • chloroplasts for photosynthesis
    • Amyloplasts for starch storage
    • Chromoplasts for pigment synthesis and storage
    • Leucoplasts – can differentiate into more specialized plastids (Amyloplasts – starch storage, Elaioplasts – storing fat, Proteinoplasts – storing and modifying protein)
    • (MH – plastids and mitochondria and have own DNA)
  • Cell Wall
    • Rigid structure outside cell membrane
    • No ability to move
    • Resist osmotic stresses
    • Structure – cellulose, hemicellulose, pectin
  • Specialized Adhesion Junctions
    • plasmodesmata
    • cell-cell communication pathways
    • allow cell membrane and endoplasmic reticulum of adjacent cells are continuous

Chloroplasts

  • disk-shaped and about 5-8 µm in diameter and 2-4 µm thick. A typical plant cell has 20-40 of them

Virus

  • not a cell Latin, virus = toxin or poison
  • not alive
  • infects living cells
  • unable to grow or reproduce outside a host cell
  • Infect different hosts (animal, plant and bacterial)
  • Classified
    • RNA or DNA viruses
    • double or single stranded

Virion

  • contains the genetic material, DNA or RNA
  • within a protective protein coat (capsid)

Bacteriophage

Prion

  • not alive
  • an infectious prion protein
  • misfolded normal protein (three-dimensional structure)
  • can form aggregates
  • Types
    • Creutzfeldt-Jacob disease (CJD) and Kuru a human neural prion disease
    • Bovine spongiform encephalopathyvery (BSE) in cattle, “mad cow disease”
    • Scrapie in sheep

Biological Levels

  • Whole cell
  • Organelles
    • nucleus, mitochondria,
  • Components
  • Biological polymers
    • chains of molecules
    • consisting of monomer subunits
    • DNA, RNA, Protein, sugars, cellulose
  • Organic molecules
    • monomer subunits
    • nucleotides, amino acids, carbohydrate

Eukaryotic Cell Organelles

  • Fundamental concept – all cells
    • Specialized exceptions
  • Organelle
  • specialized part of a cell that has its own particular function
  • Membrane bound (enclosed)
  • forms “compartments” within the cell

Chapter 11 Membrane Structure pp 363-386, Chapter 12 Membrane Transport pp 387-424, Chapter 15 Intracellular Compartments and Transport pp 495-530


Plasma Membrane

The cell membrane (plasma membrane or plasmalemma) encloses or covers all cell types and is 7 microns thick.

Compartments

  • Physical Compartments
    • membrane bound
    • Nucleus, Cytoplasm, Organelles
    • cell nomenclature based upon presence or absence of these compartments (eukaryotic, prokaryotic)
  • Functional Compartments
    • spatial localization
    • targeting
    • activation and inactivation
    • signaling

Major Cellular Compartments

  • Nucleus (nuclear) – contains a single organelle compartment
  • Cytoplasm (cytoplasmic) – contains many organelle compartments

Organelle Number/Volume

  • How many organelles?
  • How much space within the cell do they occupy?
  • Are all the cells the same?

Take a typical mammalian liver cell….

Table 12-1. Relative Volumes Occupied by the Major Intracellular Compartments in a Liver Cell (Hepatocyte)

Table 12-2. Relative Amounts of Membrane Types in Two Kinds of Eucaryotic Cells

Nuclear Compartment

  • Nuclear matrix – consisting of Intermediate filaments (lamins)
  • Nucleoli (functional compartment – localised transcription DNA of RNA genes)
  • Chromosomes (DNA and associated proteins)

Cytoplasmic Compartment

  • Cytoplasmic Organelles
    • Membrane bound structures
    • Endoplasmic reticulum, golgi apparatus, mitochondria, lysosomes, peroxisomes, vesicles
  • Cytoskeleton
    • 3 filament systems
  • Cytoplasmic “structures”
    • Ribosomes
    • DNA -> mRNA -> Protein
    • Proteins
    • Receptors, signaling, metabolism, structural
    • Viruses, bacteria, prionsl
  • Functional compartments
    • occur in nucleus, cytoplasm, in organelles and outside organelles
    • signaling, metabolic reactions, processing genetic information, cytoskeleton dynamics, vesicle dynamics

Membrane Functions

  • Form compartments
  • Allow “specialization”
  • Metabolic and biochemical
  • Localization of function
  • Regulation of transport
  • Detection of signals
  • Cell-cell communication
  • Cell Identity

Membrane Components

  • phospholipids, proteins and cholesterol
  • first compartment formed
  • prokaryotes (bacteria) just this 1 compartment
  • eukaryotic cells many different compartments

Phospholipids

  • membranes contain phospholipids, glycolipids, and steroids
  • The main lipid components include:
    • phosphatidylcholine (~50%)
    • phosphatidylethanolamine (~10%)
    • phosphatidylserine (~15%)
    • sphingolipids (~10%)
    • cholesterol (~10%)
    • phosphatidylinositol (1%).

Phospholipid Orientation

  • A liposome (lipid vesicle) is a small aqueous compartment surrounded by a lipid bilayer.
  • A micelle is a small compartment surrounded by a single lipid layer.

Membranes History

  • 1890 Charles Overton
    • selective permeation of membranes
    • non-polar pass through (lipid soluble)
    • polar refractory
    • lipids present as a coat
  • 1905 Irving Langmuir
    • lipids faced with heads towards water away from organic solvents
  • 1925 Gorter and Grendel
    • monolayer of lipid isolated from rbc
    • twice (2x) surface area of cell (bilayer)
  • 1930-40 Danielle-Davson
    • Proteins coat a bilayer with polar “pores”
  • 1960s Robertson
    • Modification with glycoprotein on one side, therefore asymmetric
  • 1972 Singer and Nicholson
    • proteins “floating” within lipid bilayer like a “liquid” surface
  • 1975 Unwin and Henderson
    • integral membrane proteins
    • both hydrophobic and hydrophilic
    • alternating -phobic and -philic represent trans-membrane loops
    • glycoprotein carbohydrate groups on outer surface

Membranes Recent History

  • 1997 Simons
    • cholesterol to form rafts that move within the fluid bilayer
    • “Membrane Rafts”
    • “A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.”

Membrane Proteins

  • 20-30% of the genome encodes membrane proteins
  • Proteins can be embedded in the inner phospholipid layer, outer phospholipid layer or span both layers
  • Some proteins are folded such that they span the membrane in a series of “loops”

Two major protein transmembrane structures

  1. α-helical – ubiquitously distributed
  2. β-barrel – outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria

Membrane Protein Functions

  • transport channels
  • enzyme reactions
  • cytoskeleton link
  • cell adhesion
  • cell identity

Membrane Glycoproteins

  • Glycoproteins are proteins which have carbohydrate groups (sugars) attached
  • to produce these proteins go through a very specific cellular pathway of organelles (secretory pathway)
  • to reach the cell surface where they are either secreted (form part of the extracellular matrix)
  • or are embedded in the membrane with the carbohydrate grouped on the outside surface (integral membrane protein)

Membrane Cholesterol

  • Small molecule embedded between the phospholipid molecules and regulates lipid mobility (MH – see rafts)
  • Cholesterol can be at different concentrations in different regions of plasma membrane
  • lateral organization of membranes and free volume distribution
  • may control membrane protein activity and “raft” formation
  • fine tuning of membrane lipid composition, organization/dynamics, function
  • bacterial membranes (except for Mycoplasma and some methylotrophic bacteria) have no sterols, they lack the enzymes required for sterol biosynthesis.

Bacterial Membranes

  • Bacteria with double membranes (Example: E. coli)
    • inner membrane is the cell’s plasma membrane
    • Gram Negative do not retain dark blue dye used in gram staining
  • Bacteria with single membranes (Example: staphylo-cocci and streptococci)
    • thicker cell walls
    • Gram Positive because they do retain blue dye
    • single membrane comparable to inner (plasma) membrane of gram negative bacteria

Membrane Specializations

  • plasma membrane cytoskeleton
  • different directly under membranes
  • adhesion complexes
  • absorbtive and secretory
  • synaptic junctions

Adhesion Specializations

A series of different types of proteins and cytoskeleton associations forming different classes of adhesion junctions (MH – covered in detail in a lecture 8)

  • Desmosomes ( = macula adherens)
  • Adherens Junctions ( = zonula adherens)
  • Septate Junctions
  • Tight Junctions
  • Gap Junctions

Membrane Transport

Three major forms of transport across the membrane

  • Passive – Simple diffusion
  • Facilitated – transport proteins
  • Active – transport proteins for nutrient uptake, secretion, ion balance

Ion Channels

  • phospholipid impermeable to ions in aqueous solution
  • protein channels permit rapid ion flux
  • 1960’s structure and function, ionophores (simple ion channels)
  • common structural motif alpha helix
  • 75 + different ion channels
  • Allosteric proteins – conformation regulated by different stimuli
  • opening/closing, “gating” of ions

Ion Channel Types

  • 3 rapid + 1 slow gate (gap junction)
    • Voltage-gated – propagation of electrical signals along nerve, muscle
    • Ligand-gated – opened by non-covalent, reversible binding of ligand between nerve cells, nerve-muscle, gland cells
    • Mechanical-gated – regulated by mechanical deformation
    • Gap junction – allow ions to flow between adjacent cells open/close in response to Ca2+ and protons

Apoptosis and Membranes

  • programmed cell death
  • membrane “blebbing” encloses cellular component fragments

Membrane Transport Disease

  • Cystic Fibrosis
    • 1989 Collins (US), Tsui and Riordan (Canada)
    • Chloride channel protein mutation
    • point mutant, folded improperly, trapped and degraded in ER

Chapter 5 DNA and Chromosomes pp 171-196


Cell Nucleus

This lecture introduces the nucleus and how information is transferred from stable stored information (DNA) converted to an intermediate (mRNA, rRNA, tRNA) of variable stability, exported from the nucleus to the cytoplasm where mRNA is then translated into Protein. This is gene expression, the products of this process are used either within the cell, exported (exocytosis) or used to replace worn out components.

We will study this topic looking at the key organelle in this process, the nucleus.

Eukaryotes

Difference between Prokaryotes and Eukaryotes

  • Cytoskeleton
  • DNA structure
    • circular, linear
    • Packing (histones)
    • RNA processing (splicing)

Eukaryote Gene Expression

DNA -> mRNA -> Protein

DNA (transcription) -> mRNA (translation) -> Protein (function)

Nucleus

DNA (transcription) -> mRNA Nuclear processing (export)

  • DNA -> mRNA splicing (introns removed, exons joined) -> mRNA

Cytoplasm

mRNA (translation by ribosomes) -> Protein (processing)

  • Protein Processing cytoplasm (free ribosomes), rough endoplasmic reticulum (bound ribosomes)

Membrane Evolution

Postulated that an early “coating” structure lead to the infolding of the primitive plasma membrane to form the many membrane covered organelles in the cytoplasm.

These modules may also be the evolutionary precursor to the nuclear pore structures and account for the double membrane that coats the nucleus.

Nuclear Compartment

  • Nuclear envelope
  • Nuclear cytoskeleton
  • Nucleolus
  • Chromosome territories
  • Interchromatin compartment
  • “speckles” interchromatin granule clusters
    • Splicing speckles or SC 35 domains
    • thought to be sites of storage of mRNA splicing factors
  • nuclear bodies – Cajal and PML

Nucleus Size

  • cell “karyoplasmic ratio” relatively constant (ratio of nuclear volume to cell volume)
    • most other cellular organelles (ER and mitochondria) can vary greatly in amounts
  • multinucleated fission yeast cells
    • relative amount of cytoplasm surrounding each nucleus controls the size of individual nuclei

Nuclear Envelope

  • Forms structural compartment
  • Nuclear envelope two concentric membranes
  • Breaks down each mitosis (recycled)
  • Outer membrane continuous with Endoplasmic Reticulum (Endoplasmic Reticulum is covered in Lecture 5)
  • Contains holes “nuclear pores”

Nuclear Cytoskeleton

The nuclear cytoskeleton has 2 layers

  • outer – less organised surrounds membrane
  • inner – nuclear lamina – thin shell (20 nm) underlying the membrane (nuclear envelope)
  • associates with both the inner nuclear membrane and underlying chromatin
  • can regulate gene expression
  • provides anchor sites for nuclear pore complexes
  • broken down each cell division

Nuclear Lamins

  • intermediate filaments
  • large family of different filament types (covered in Cytoskeleton Lecture – Intermediate Filaments)
  • 10 nm in diameter, forms rope-like networks
  • lamins, Class V intermediate filaments
    • polypeptide form dimers
    • central alpha-helical regions of two polypeptide chains are wound around each other
  • assembly
    • head-to-tail association of dimers form linear polymers
    • side-by-side association of polymers form filaments
  • B-type lamins are ubiquitously expressed throughout development
  • A-type lamins in many organisms expression does not appear until midway through embryogenesis (possible role in differentiation)
  • Lamin Abnormalities – laminopathies

Nuclear Pores

  • Protein complex
  • External diameter of about 120 nm (30 times the size of a ribosome)
  • Channel diameter 25 nm
  • channels between nucleus and cytoplasm (import/export)
  • passive passage of small polar molecules, ions,
  • active (selective/ regulated) passage of macromolecules, proteins and RNAs

Nuclear Bodies

  • also called – nucleolar accessory bodies, coiled body, gems
  • 0.1 – 2.0 microns, 1-10/ nucleus
  • Gems and Cajal bodies two forms of same structure
  • GEMS (Gemini of coiled bodies)
  • proposed sites where small nuclear ribonucleoproteins (snRNPs) and small nucleolar RNAs (snoRNPs) are modified.
    • snRNPs are particles that combine with pre-mRNA and various proteins to form spliceosomes
    • snoRNAs are a class of small RNA molecules that are involved with modifications of ribosomal RNAs (rRNAs) and other RNA genes

Chromosomes

  • not “visible” at interphase, condense for mitosis (1,000 fold)
    • condensation allows chromosomes to move along mitotic spindle without breaking or tangling
  • eukaryotes have separate chromosomes
    • Human 23 pairs, 22 autosome pairs, 2 sex chromosomes
  • diploid 2 copies of each chromosome (inherited one male/one female)
    • except male sex chromosomes X from mother Y from father
  • DNA and protein
  • packing of DNA
  • DNA structure
  • encodes genome (humans 30,000 genes, draft sequence published in 2001)
  • DNA genes encode RNA and proteins
  • DNA can also encodes nothing

Chromosome Territories

  • Space within the nucleus occupied by individual chromosomes
    • Several different models as to how these territories interact
  • Intrachromosomal domains possibly RNA processing and transport

Nucleolus

  • Appearance
    • Fibrillar center, dense fibrillar component, and granular component
    • Nucleolus changes during the cell cycle:
      • during mitosis – nucleolus breaks up as chromosomes condense
      • after mitosis – nucleolus reforms from coalesce of tips of 10 chromosomes
  • Function
    • Sites of ribosomal (rRNA) gene transcription, processing, and ribosome assembly
    • Nucleolus size depends on cell metabolic activity
    • Sites of ribosomal (rRNA) gene transcription, processing, and ribosome assembly
    • All cells contain multiple copies of rRNA genes

Chromosome Features

  • 2 telomeres, centromere, replication origins
  • Telomere- at ends of chromosome (bacterial DNA circular)
  • Centromere- holds duplicated DNA together
  • Kinetochore – protein complex forms around the centromere forms during mitosis
  • Chromatin – DNA packed by DNA binding proteins (histones and non-histones) form 30nm DNA fibre
  • 2 types of chromatin in interphase nuclei (based on cytology)
    • heterochromatin – highly condensed (restricted gene transcription)
    • euchromatin – less condensed (gene transcription)

Telomere

  • at ends of all chromosomes (not bacterial DNA circular)
  • roles in chromosome replication and maintenance
  • replication
    • for replicating the ends of linear chromosomes
  • maintenance
    • proposed to provide each cell with a replication counting mechanism that helps prevent unlimited proliferation
  • each cell division shortens telomere 50–100 nucleotides
  • DNA 100s to 1,000s repeats of a simple-sequence containing clusters of G residues (humans AGGGTT)
  • Telomerase enzyme maintains length

Centromere

  • directs movement of each chromosome into daughter cells every time a cell divides
  • centromere embedded in heterochromatin
  • satellite DNA sequences (AT-rich) repeated many thousands of times
  • proteins assemble on this to form Kinetochore
    • attachment site for spindle microtubules

Replication Origins

  • DNA replication initiates at multiple origins (ori)
  • in both prokaryotic and eukaryotic DNA
  • multiple origins in eukaryotes (human genome about 30,000 origins)
  • each origin produces two replication forks (moving in opposite directions)

Chromosome DNA Packing

Nucleosomes

  • formed by DNA wrapped around histones
  • unit particle of chromatin (nucleosomal histones) (discovered 1974)
  • EM unfolded DNA has “beads on a string” appearance
  • second order folding forms 300 nm fibre
  • condensed DNA for mitosis 700 nm fibre

Histones

  • only in eukaryotes
  • small proteins positively charged (binds negatively charged DNA)
  • not sequence specific binding (as in transcription factors)
  • 4 core histones (H2A, H2B, H3, and H4)
  • 2 linker histones (H1/H5)

Eukaryote Gene Expression

DNA -> mRNA -> Protein

  • DNA (transcription) -> mRNA (translation) -> Protein (function)
  • DNA -> mRNA splicing (introns removed, exons joined) -> mRNA
  • DNA -> rRNA, tRNA, siRNA (RNA interference (RNAi) pathway

Nucleus DNA (transcription) -> mRNA Nuclear processing (export) Cytoplasm mRNA (translation) -> Protein (cytoplasm, rough endoplasmic reticulum)

Protein Modification

Protein – cytoplasmic (free ribosomes), rough endoplasmic reticulum (bound ribosomes)

Exocytosis

Rough Endoplasmic Reticulum -> transport vesicle -> Golgi apparatus -> secretory vesicle

Cell Export – Exocytosis


Cell Export – Exocytosis

Introduction

This lecture introduces how information is transferred from stable stored information (DNA) converted to an intermediate (mRNA, rRNA, tRNA) of variable stability, exported from the nucleus to the cytoplasm where mRNA is then translated into Protein. This is gene expression, the products of this process are used either within the cell, exported (exocytosis) or used to replace worn out components.

We will study this topic at the level of the cellular components and organelles involved in the process: ribosomes, endoplasmic reticulum, Golgi apparatus, vesicles (transport and secretory).

Looking in the Cytoplasm

Difference between Prokaryotes and Eukaryotes

  • Light microscope – histology, immunohistochemistry
    • lacks details within cytoplasmic compartment
    • Immunochemistry
    • Organelle dyes
    • Fluorescent tagged proteins
  • Electron microscope
    • shows the organelles and membrane structure

Links: MCB – The secretory pathway of protein synthesis and sorting. | MCB – movie – Protein Secretion

The Cytosol

  • Membrane bound compartment
  • About 1/2 total cell volume
  • Intermediary metabolism takes place in the cytosol
    • Chemical biological reactions
    • Degradation
    • Synthesis
  • Protein molecules
    • cell has about 10 billion (1×1010)
    • 10,000 to 20,000 different kinds

Compartments are Dynamic

  • Membrane bound compartments change shape and size
  • Related to cell cycle, differentiation, signaling

Links: MCB – Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells | MCoB Table 12-3. Some Typical Signal Sequences

Ribonucleic Acid (RNA)

  • 3 types of RNA
    • Messenger RNA (mRNA) translated into protein by action of ribosomes
    • Transfer RNA (tRNA) each tRNA is specific for a specific amino acid (anti-codon)
    • Ribosomal RNA (rRNA) Forms the backbone of ribosome subunits

Messenger RNA (mRNA)

  • processed in the nucleus
  • biological molecule (polymer) with unstable (half-life)
  • exported to the cytoplasm
  • site of ribosome assembly

Messenger RNA movie

Transfer RNA (tRNA)

  • small RNA molecules which each binds a specific amino acid (anti-codon) tRNA
  • carries it to the ribosome for protein assembly

Ribosomal RNA (rRNA)

  • provide framework for dozens of proteins involved in assembly of AA sequence into protein
  • most abundant RNA in cells
  • Usually characterized by sedimentation coefficient
    • 40S & 60S
  • rRNA genes are located in nucleolus
  • multiple copies of human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. PMID: 3336775

Links: MCB – Overview of mRNA processing in eukaryotes | MCB – movie – Life Cycle of an mRNA

Ribosomes

  • 1955 A small particulate component of the cytoplasm. PALADE GE. J Biophys Biochem Cytol. 1955 Jan;1(1):59-68. PMID: 14381428
  • 2009 The Nobel Prize in Chemistry 2009 awarded to Drs Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath “for studies of the structure and function of the ribosome”.

Cartoon animation of translation on ribosome and export into endoplasmic reticulum

Ribosome Structure

  • Ribosome biogenesis consumes up to 80% of the energy of the cell PMID: 10806485
  • All RNA components mRNA, rRNA and tRNA come together in this structure
  • two ribosome types with identical structure
  • different locations – free and membrane bound
    • Free in cytoplasm
    • Bound to endoplasmic reticulum
  • Also located within mitochondria

Ribosome Function

  • Protein Synthesis
  • complexes where RNA sequences are converted to amino acid (aa) sequences
  • Codons 3 NTPs = 1 AA
    • AA incorporated at 20/sec
    • average sized protein takes 20-60 seconds to assemble
  • Synthesis from amino- to carboxy- terminal of protein
  • many ribosomes can bind 1 mRNA (polyribosome)

Polyribosomes

  • polyribosomes or polysomes are the EM visible granules
  • many ribosomes bound to a single mRNA
  • single ribosome covers a 54bp mRNA region
  • the synthesised single amino acid chain can then be “modified”
    • in the cytoplasm or in specialised organelles
  • Protein Modification/Function

Links: MBoC – Figure 1-10. A ribosome at work | MBoC – Figure 12-37. Free and membrane-bound ribosomes | MBoC – Figure 6-63. A comparison of the structures of procaryotic and eucaryotic ribosomes |MCB – Model of protein synthesis on circular polysomes and recycling of ribosomal subunits | MCB – movie ch4anim4.mov

Endoplasmic Reticulum

  • endoplasmic ‚”within the cell
  • reticulum ‚ “a little net
  • an organelle, membrane bound compartment. within the cytoplasmic space
  • One structural compartment
  • Two functional compartments
    • Rough Endoplasmic Reticulum (RER)
    • Smooth Endoplasmic Reticulum (SER)

Rough Endoplasmic Reticulum – Function

Mammalian proteins transported into er

  • Allows specific proteins to be modified and targeted to different destinations
  • Modification
    • amino acid chain cleaved or sidegroups added (mainly glycosylation)
    • glycosylation = addition of carbohydrate (sugar) groups
  • Destination
    • Domestic – Cytosolic, Nuclear, Organelles
    • Exported from cell

Links: MCB – Overview of sorting of nuclear-encoded proteins in eukaryotic cells | MCB – movie – Protein Sorting JCB- movie – Real-time video of the formation of tubules at ER export sites

Rough Endoplasmic Reticulum – Structure

  • about 50% of cell membrane
  • continuous with outer nuclear membrane
  • single highly convoluted membrane enclosing a single space
  • ER lumen = ER cisternae
  • “rough” because of many ribosomes attached to the membrane
  • ribosomes bound only to cytoplasmic side of ER membrane

Smooth Endoplasmic Reticulum – Structure

  • Part of same membrane as RER
    • may also be called “transitional”
  • no attached ribosomes
  • not involved in protein synthesis
  • differ in shape
  • SER a meshwork of fine tubules

Smooth Endoplasmic Reticulum – Function

  • lipid metabolism (membrane)
  • carbohydrate metabolism
  • detoxification of drugs and harmful compounds
  • steroid synthesis and metabolism (cholesterol)
  • different amounts in different cells

In muscle cells SER stores and releases calcium to trigger muscle contractions.

Links: MBoC – Transport from the ER through the Golgi Apparatus

Transport Vesicles

  • RER synthesized material is transferred by budding off of membrane
  • Forms transport vesicle
  • Transports substances to different cellular locations
  • Most transport to Golgi apparatus
  • Active transport mainly along microtubules (cytoskeleton)

Links: MBoC – Vesicular Traffic | JCB – movie – transport vesicles and lipid (large 9.7 Mb)

Golgi Apparatus

Links: MBOC – Golgi Apparatus- Summary

Golgi Apparatus – Structure

  • organelle, membrane enclosed structural compartment
  • cell may contain one or more Golgi apparatus
  • located near the nucleus
  • disc shaped membrane stack with different regions by their location within the cell

Golgi stack

  • from 6-30/stack
  • 3-100s stacks/cell
  • many sets of membrane bound smooth surfaced cisternae

Stack Nomenclature

  • cis – bottom of stack closest to endoplasmic reticulum, receives transport vesicles from ER
  • medial – middle of stack, processing of proteins, modification of sidechains
  • trans – top of stack closest to plasma membrane, buds off secretory vesicles

Links: MBoC – Golgi Apparatus | MBoC – Figure 13-30. Two possible models explaining the organization of the Golgi apparatus and the transport of proteins from one cisterna to the next | MCB – Figure 5-49. Three-dimensional model of the Golgi complex built by analyzing micrographs of serial sections through a secretory cell

Golgi Apparatus – Functions

  • Sorting of cytosolic/secreted proteins
  • Glycosylation of secreted proteins
  • Modification of carbohydrates
  • Side chains are also trimmed
  • Trans vesicles fuse with the plasma membrane

Secretory Vesicles

  • protein export (secretion)
    • constitutive and regulated
  • related also to membrane turnover
    • new lipid
    • new cholesterol
    • new membrane proteins

Chapter 15 Cell Import – Endocytosis pp 522-529


Cell Fractionation Techniques

  • Centrifugation generated 4 fractions
    • Nuclear
    • Mitochondrial
    • Microsomal
    • Supernatant

Links: MBOC – Cell fractionation by centrifugation

Cytosolic Vesicles

  • Single membrane bound vesicles
  • two membrane processes involved in trafficking
    • budding
    • fusion
  • Linked to the ER and Golgi membrane system
    • Lysosomes
    • Peroxisomes

Peroxisomes

  • organelles that metabolize fatty acids
  • increased activity of digestion in enzyme studies
  • Identified by EM 10 years later
  • enzymes that produce and others that degrade hydrogen peroxide (a reactive oxygen species, ROS)
    • oxidative reactions using molecular oxygen to generate hydrogen peroxide
    • oxidizing fatty acids, bile salts and cholesterol
    • then converting hydrogen peroxide to nontoxic forms
  • Catalases (EC 1.11.1.6)
    • haem-containing proteins that catalyse conversion of hydrogen peroxide (H2O2) to water and molecular oxygen

Peroxisome Assembly

  • Two theories on peroxisome formation
  • semiautonomous oranelles (like mitochondria) which multiply strictly by growth and division
    • Free ribosomes synthesize peroxisome proteins
    • Imported into pre-existing peroxisomes as completed polypeptide chains
    • Peroxisome growth from protein import
    • formation of new peroxisomes by division of old ones
  • other organelles such as ER role in formation and maintenance of peroxisomal membranes

Links: MBOC – A model for how new peroxisomes are produced | Lippincott-Schwartz Lab

Absorption Mechanisms

Endocytosis Types

Receptor Mediated Endocytosis

  • General term for all mechanisms of absorbtion extracellular fluid and substances
  • substances bind to receptor sites
  • vesicle called endosome
  • can be utilized by viruses to enter cells

Pinocytosis

  • “cell drinking”
  • All cells, extracellular fluid
  • micropinocytosis within vesicles (<0.1 µm diameter)
  • macropinocytosis within vacuoles (0.5-5.0 µm) named macropinosomes

Phagocytosis

  • “cell eating”
  • occurs only in specialized cells macrophages, dendritic cells and neutrophils
  • capture and destroy pathogens and particulate antigens
  • essential component of the innate immune response
  • Fc- and complement-receptor mediated phagocytosis, named for binding specificity for antibody tail region called Fc (Fragment, crystallizable)
  • clearance of apoptotic bodies
  • some bacteria “hijack” this process in non-phagocytic cells to enter and infect them

Endosome

  • vesicle formed from plasma membrane budding
  • encloses extracellular fluid and substances
  • large ones called a phagosome or vacuole

Clathrin-coated Pits

  • Clathrin is a protein that coats both small membrane pits and coated vesicles
    • formed during endocytosis of materials at the surface of cells
  1. macromolecules to be internalized bind to specific cell surface receptors
  2. receptors are concentrated in regions of the plasma membrane (clathrin-coated pits)
  3. pits bud from the membrane to form small clathrin-coated vesicles containing the receptors and bound macromolecules

Autophagy

  • lysosomal degradation pathway for cytoplasmic material
  • survival mechanism during short-term starvation

Lysosomes

The Cell – Endocytosis and lysosome formation

  • Are the site of cellular digestion
    • contain up to 40 enzymes for digestion
  • Acid Hydrolases
    • Active at acid pH (5)
  • Hydrogen ion pump in lysosomal membrane
    • drives ions from cytoplasm into lumenal space
    • generates internal acidic environment

Lysosome Membrane

  • Allows passage of uncharged molecules
  • Molecules enter, are charged and cannot leave

Lysosome Digestive Enzymes

  • Acid hydrolases
  • enzymes named on basis of substrate
    • Protease – digests proteins
    • Nuclease – digests neucleotides (DNA)
    • Glycosidase – digests carbohydrates (sugars)
    • Lipases – digests lipids (fats)
    • Phospholipases – digests phospholipids (membranes)
    • Phosphatases – removes a phosphate group

Lysosome Types

  • Primary
    • newly formed without digestive substrate
    • formed from budding Golgi apparatus
    • can be secreted by exocytosis
  • Secondary
    • active form enzyme + substrate
    • formed by vesicle fusion event
  • primary lysosomes in neutrophils are called primary granules or A granules

Caveolae

  • small membrane invaginations
  • defined by containing caveolin protein in the vesicle membrane
  • not always present in all cells
  • functions
    • lipid recycling
    • cellular signalling
    • endocytosis

Transport Vesicles

  • RER synthesized material is transferred by budding off of membrane
  • Forms transport vesicle
  • Transports substances to different cellular locations
  • Most transport to Golgi apparatus
  • Active microtubule-based transport
    • also may use microfilament transport

Endoplasmic Reticulum and Golgi

  • Both these systems involved with both cell import and export
    • New proteins synthesized on membrane-bound ribosomes are transported through the Golgi apparatus
    • reach the trans-Golgi network (TGN) and sorted for delivery to various destinations
    • exocytosis and endocytosis pathway
  • The question is how these compartments “sort” components going in different directions?
  • biodigested products from the digestive lysosomal pathway need now to be delivered to the biosynthetic pathway
    • amino acids, nucleotides, carbohydrates, phospholipids, lipids, etc

Other Vesicles

Exosomes

  • nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types
  • Function suggested as the eradication of obsolete proteins, antigen presentation, or “Trojan horses” for viruses or prions. (PMID: 16809645)

Endosomal Multivesicular Bodies (MVBs)/endosomes

  • a stage in endosomal development
  • A type of cytoplasmic vesicle (200 – 500 nmdiameter) that occurs when part of an endosome membrane invaginates and buds into its own lumen forming smaller contained vesicles.
  • smaller contained vesicles are degraded when the endosome fuses with a lysosome.
  • allows delivery of transmembrane proteins into the lumen of the lysosome for degradation.
  • compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. (PMID: 12892785)
  • delivery of transmembrane proteins into the lumen of the lysosome for degradation is mediated by the multivesicular body pathway. (PMID: 15569240)
    • The ESCRT (ESCRT-I, -II and -III) complexes form a network that recruits monoubiquitinated proteins and drives their internalization into lumenal vesicles within a type of endosome known as a multivesicular body. (PMID: 16689637)
  • essential for both sorting and multivesicular endosomes formation (PMID: 12892785)

Macropinosomes

  • Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures.

Phagosomes

  • fusion of endoplasmic reticulum (ER) with macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages
  • phagocytic cup
    • actin-based membrane structure formed at the plasma membranes
    • impaired in Wiskott-Aldrich syndrome (WAS)

Synaptic Vesicles

  • secreted vesicle
  • neuron specific
  • filled with neurotransmitter

Melanosomes

  • membrane vesicle enclosing melanin
  • melanin is a light-absorbing pigment
  • skin melanocytes and retinal pigment epithelium cells
  • melanophages are cells that engulfed the released melanosomes (eg skin keratinocytes)
    • skin colour due to melanocytes level of activity not to the number of melanocytes

Proteasomes

  • a protein complex that degrades proteins by proteolysis
    • misfolded, unneeded or damaged proteins
  • not vesicles, proteins form a “stacked-ring” structure
  • proteins destined for destruction are ubiquitinated (ubiquitination)
    • attachment of one or more ubiquitin monomers to protein

Chapter 14 Energy Generation in Mitochondria and Chloroplast pp453-492


Double Membrane Organelles

  • Nucleus – all eukaryotes
  • Chloroplasts – plants
  • Mitochondria – plants and animals

Evolution Mitochondria

  • primitive Eubacterium
  • symbiotic relationship with eukaryotic cell
    • circular DNA
    • see antibiotic-induced deafness due to similarity of mitochondrial and bacterial ribosomes
  • genes transferred to nucleus

Chloroplasts

  • Plant Chloroplast organelles
  • Double membrane cytoplasmic organelle
  • present in photosynthetic Eubacteria, algae and plants
    • thought to originate as an endosymbiotic cyanobacteria (blue-green algae)

Function

  • photosynthesis
  • chlorophyll captures light energy
  • chloroplasts interact with peroxisomes

Structure

  • flat discs usually 2 to 10 micrometer in diameter and 1 micrometer thick. In
  • plants 5 μm in diameter and 2.3 μm thick
  • inner and an outer phospholipid membrane
  • intermembranous space
  • stroma
    • stacks of thylakoids (site of photosynthesis)
    • contains copies of small circular DNA
    • ribosomes
    • proteins transported to the chloroplast

(MH – will not cover this cell organelle in any depth in current course)

Mitochondria

  • Located throughout cytoplasmic compartment
    • has itself several membrane enclosed compartments
    • each compartment has different function
  • Ancient aerobic organisms in symbiosis (endosymbiosis)
  • present in all cells

Mitochondria Function

  • Energy production
    • Respiratory chain
  • Signaling
  • Apoptosis role
    • Programmed cell death

Mitochondria Structure

  • Double membrane
  • outer membrane
  • intermembrane space
  • inner membrane
  • crista (plural, cristae)
    • originally considered specialized folds of the inner membrane
    • variable invaginations with narrow tubular connections to each other and by crista junctions to the peripheral region of inner membrane
  • matrix

Mitochondria Shape

  • Come in different shapes & sizes
  • Can rapidly change shape (minutes)

Mitochondria Location

  • cells with high energy requirements: Muscle, sperm tail, flagella
  • generally located where energy consumption is highest in the cell
  • Mitochondria (fibroblasts)
  • Mitochondria (sperm)
    • Packed around initial segment
    • Energy for sperm motility, microtubules (9+2)

Mitochondria Components

Outer Membrane

  • porin – membrane channel, allows ions and metabolites into the mitochondria (<5000 daltons)

Intermembrane Space

  • similar to the cytosol with respect to the small molecules it contains
  • also enzymes that use ATP

Inner Membrane

  • cardiolipin – phospholipid, makes membrane impermeable to ions
  • transport proteins – permeable to molecules required in the matrix

Cristae

  • increase inner membrane surface area
    • tubular, vesicular or flat cristae
  • Adenosine triphosphate (ATP) synthase
  • respiratory electron transfer chain proteins
  • transport proteins

Matrix

  • metabolic enzymes of citric acid cycle (=Krebs) (100s of enzymes) (MH– do not need to know biochemical details of this cycle)
  • genetic material DNA, tRNA, ribosomes

Mitochondria DNA

Eukaryotic mitochondrial genomes

  • double stranded circular DNA (mitoDNA. mtDNA)
  • 1981 complete human sequence (16,569 nucleotides)
  • 37 genes
    • encodes 13 polypeptides involved in oxidative phosphorylation
    • remaining genes transfer RNA (tRNA) and ribosomal RNA (rRNA)
  • multiple copies within the matrix
  • maternally inherited
  • remainder encoded by nuclear DNA
  • proteins made in cytosol and imported into mitochondria

Links: Home Reference – Mitochondrial DNA

Mitochondria Protein Synthesis

Many mitochondrial proteins are encoded by nuclear DNA

  • synthesis begins in the cell cytoplasm
  • imported into the mitochondria
    • targeting similar to signal sequence for RER
  • once in matrix signal sequence is cleaved (by Hsp70)
    • protein then folds (by Hsp60)
  • proteins for mitochondrial membrane or intermembranous space
  • additional signal following matrix localization

Mitochondrial targeting signal (MTS) – alternating amino acid pattern (amphipathic helix) with a few hydrophobic amino acids and a few plus-charged amino acids at the N terminus.

Mitochondria Fission

  • Mitochondrial Division
  • Divide independently of the whole cell cycle
  • Generated by existing mitochondria
  • inward furrowing like bacterial division
    • mitochondria lack FtsZ ring (seen in bacteria)
    • rely on dynamin on the cytosolic face for fission

Mitochondrial Fusion

  • when two separate mitochondria join as one
  • fission and fusion considered to be balanced
  • disruption causes normal tubular network of mitochondria to fragment into short rods or spheres

Energy Production

Respiration

  • Raw Materials
    • Oxygen
    • Pyruvate & Fatty Acids
  • Products
    • Carbon Dioxide
    • Adenosine Triphosphate (ATP)

Apoptosis

Mitochondria in addition to energy production, have a second major function related to programmed cell death by apoptosis.

  • cytochrome C release activates caspases
  • other changes include
    • electron transport, loss of mitochondrial transmembrane potential
    • altered cellular oxidation-reduction
    • Bcl-2 family proteins (pro- and antiapoptotic)
  • Vesicular Mitochondria
    • begin to appear during the release of cytochrome C which initiates mitochondrial mediated apoptosis
    • transformation from normal morphology
    • with an inner boundary membrane connected to lamellar cristae via crista junctions
    • multiple vesicular matrix compartments
    • facilitates membrane fission or fragmentation as the matrix is fragmented at this stage
    • fragmentation of the mitochondrion requires only outer membrane fission

(MH- this topic will be covered again in the Cell Death Lecture)

Nuclear transfer of mitochondrial DNA

  • mitochondria to the nucleus generates nuclear copies of mitochondrial DNA (numts)
  • Integration can appear as neutral polymorphism or associated with human diseases (insertion of mtDNA into genes,5 known cases), the mitochondrial genome remains intact in the individuals.

Leber Hereditary Optic Neuropathy

  • maternally inherited cause of blindness Genes and Diseases – LHOM
  • mutation of mitochondrial DNA (mtDNA)
    • three common mtDNA mutations: G11778A, T14484C, G3460A

Chapter 20 Cellular Communities:Tissues, Stem Cells, and Cancer – Epithelial Sheets and Cell Junctions pp700-705


Why Adhesion?

  • Adhesion refers to “stickiness”
  • Evolution of multicellular organisms developed specialized cells and tissues
  • Embryonic development also allows differentiation of different cell/tissue types
  • Interaction between cell-cell and cell-extracellular matrix by specific contacts
  • Note the Cell Biology definition is different from the Clinical term
    • Clinical term “adhesions” bands of scar-like tissue forming between two surfaces inside the body

Prokaryotes

  • Prokaryotes adhesion molecules usually termed “adhesins”
  • occur on pili (fimbriae), flagellae, or the cell surface
  • adhesion first step in colonization

Unicellular Eukaryotes

  • express multiple adhesion molecules for nutrition, migration and pathogenesis
  • malarial parasite (Plasmodium falciparum) uses circumsporozoite protein, an adhesion molecule, to bind to liver cells
  • merozoite surface protein to bind red blood cells

Multicellular Eukaryotes

  • Maintains body form and structure
  • Tissues organized during development
  • Can be used for cell migration
  • Cell signalling Alteration in disease

Types of Adhesion

  • Cell-cell
  • Cell-extracellular matrix

Adhesive Functions

  • Basal lamina assemble and organize epithelia
  • Smooth muscle
    • Maintains integrity during contraction
  • Binds growth factors
    • Neurons growth cone guidance, fasiculation
  • Cell Migration
    • Development – migration, cell sorting, tissue development
    • Transmigration, wound healing, macrophages

Adhesion Characteristics

  • Transmembrane glycoproteins
  • Normally permanent
  • Except migrating cells and embryonic
  • Changes with development
  • Loose adhesion when mature or disease
  • Erythrocytes, cancer

Types of Adhesion Molecules

  • Cadherins
  • Immunoglobulin Superfamily
  • Selectins
  • Gap Junctions (Connexins)
  • Integrins

Cadherins

  • The cadherin superfamily comprises classical and non-classical cadherins
    • present in all multicellular animals
    • mediate Ca2+ dependent cell-cell adhesions
    • more than 180 members in humans
  • Classical cadherins (e.g.: E-cadherin, N-cadherin and P-cadherin) contain 5 cadherin repeats
  • Require calcium ions to bind
  • Homophilic binding through end element
  • Like with like
  • Functional unit a dimer
  • Non-classical cadherins (e.g. desmosomal cadherin, protocadherins and T-cadherins) are more distantly related in sequence
  • Varying number of cadherin repeats
  • Some non-classical cadherins have primarily a signaling function

Immunoglobulin Superfamily

Vertebrates have 100+ In addition to adhesion they also have role in immune system Contain varying number of Ig-related domains

Selectins

Cell Surface carbohydrate-binding proteins

Vertebrates have only in circulatory system Role in inflammatory response: adhesion of leukocytes (blood cells) to endothelium (vessel wall)

Cooperate with integrins and Ig-SF receptors Selectins 2 Heterophilic interactions Bind counterreceptors

  • L-selectin on white blood cells
  • P-selectin on blood platelets and on endothelial cells that have been locally activated
  • E-selectin on activated endothelial cells

Integrins

  • Mammals have genes for 18 alpha and eight beta integrins
  • Role in cell adhesion to extracellular matrix (ECM) basement membranes
  • Induction of cell polarization by adhesion
  • Cell migration through ECM will discuss in ECM lecture
  • Glycosylated proteins
  • Bind through C terminal lectin domain of selectin
  • Comprising sandwich of beta sheets
  • Held together by hydrophobic interactions
  • Mainly receptors for ECM proteins
  • Fibronectin, laminin, collagen
  • Some heterotypic binding Ig superfamily
  • Interact with cell cytoskeleton
  • key component in signalling

Cell Junction Types

  • Desmosomes (macula adherens)
  • Adherens Junctions (zonula adherens)
  • Septate Junctions
  • Tight Junctions
  • Gap Junctions
  • Tunneling nanotubes

Desmosomes

  • intermediate filaments anchor the dense plaque that occurs under the membrane of each cell
  • desmos = bond
  • skin, lining of internal body cavity surfaces
  • disappear when cells are transformed

Hemi-Desmosome

  • cell anchored to extracellular matrix
  • Present in tissues subject to shear or lateral stress
  • Hemi=half

Adherens Junctions

  • microfilaments anchor the plaque that occurs under the membrane of each cell
  • plaques not as dense also occur as hemiform
  • heart muscle, layers covering body organs, digestive tract
  • transmembrane proteins
  • Cadherin

Tight Junctions

  • Discovered by M.G. Farquhar and G.E. Palade in 1963
  • zonula occludens
  • Fusion of 2 plasma membranes acts as a “seal”
  • Epithelia lining
  • digestive system gut, ducts, cavities of glands, liver, pancreas capillary walls urinary bladder

Gap Junctions

  • allowing direct communication between cells (open & close)
  • connexins form hollow 1.5 nm diameter cylinders
  • heart muscle, smooth muscle electrical and chemical integration as a single functional unit
  • Also in embryonic development
  • two hemichannels (connexons)
  • each formed from 6 connexin molecules

Tunneling nanotubes

  • allowing direct communication between cells
  • connecting cells at a distance of up to several cell diameters
  • tubes with a diameter of 50-200 nm

Junctions Overview – Typical Epithelia

  • Tight Junction
    • seals neighbouring cells
  • Adherens Junction
    • joins actin bundles between cells
  • Desmosome
    • joins intermediate filaments between cells
  • Gap Junction
    • cell-cell communication, passage of small molecules
  • Tunneling nanotubes
    • cell-cell communication, passage of organelles
  • Hiemidesmosome
    • anchors cell intermediate filaments to the basal lamina (extracellular matrix)

Extracellular Matrix

Substances secreted by cells lying outside the cell membrane Exocytosis

Integrins

Mammals have genes for 18 alpha and eight beta integrins Role in cell adhesion to extracellular matrix (ECM) basement membranes Induction of cell polarization by adhesion Cell migration through ECM Mainly receptors for ECM proteins Fibronectin, laminin, collagen Some heterotypic binding Ig superfamily Interact with cell cytoskeleton signalling

Focal Adhesions

  • links the outside of the cell (ECM) through transmembrane proteins (integrins) with the cell cytoskeleton (actin microfilaments) extracellular matrix integrins actin cytoskeleton

Chapter 17 Cytoskeleton pp 571-608


Function

  • functions based upon the filaments physical properties
    • each filament system has different properties
  • integral strength
  • cell shape
  • motility
    • inside the cell
    • whole cell
    • motor proteins associated with 2 filament systems
  • signal transduction

Structure

  • Network of filamentous proteins
    • filaments formed from a few proteins
    • monomer protein forms polymer filaments
  • located in nucleus and cytoplasmic compartments
    • not within organelles
  • location based upon cellular function
  • named on basis of physical size

DNA (transcription) -> mRNA Nuclear processing (export)

  • DNA -> mRNA splicing (introns removed, exons joined) -> mRNA

Alternative Splicing of pre-mRNA allows the econding of multiple mRNAs by one single gene.[1]

  • some examples are:
    • lamins (nuclear intermediate filaments)
    • tau (mictotubule-associated proteins)
    • tropomyosins (actin-associated proteins)

Organization

  • cytoplasmic
    • cortical meshwork under plasma membrane
    • three dimensional meshwork through cytoplasm
  • nuclear
    • cortical meshwork under nuclear envelope
  • assembly
    • some spontaneous
    • assembly sites
  • dynamic
    • variable stability
    • high to low stability
    • stability can be altered by associated proteins and signals
    • drugs can alter stability

Cytoskeleton Filaments

Microfilaments

  • Twisted chain 7 nm diameter
  • most abundant protein in cells (5% of all cell protein)
  • actin 43 Kd
  • Motility
  • Adhesion, focal adhesions
  • Actin binding proteins
  • myosin motors
  • Muscle actins

Intermediate Filaments

  • different cell types, different intermediate filaments
    • all eukaryotes nuclear cytoskeleton the same
  • resist stresses applied externally to the cell
  • cytoplasmic
  • anastomosed network
  • flexible intracellular scaffolding
  • 10-nanometer diameter
  • cross-linking proteins allow interactions with other cytoskeletal networks
  • intermediate filament associated proteins (IFAPs)
    • coordinate interactions between intermediate filaments and other cytoskeletal elements and organelles,
  • human disorders
    • mutations weaken structural framework
    • increase the risk of cell rupture

Microtubules

  • 25 nm diameter, 14 nm internal channel
  • tubulin
  • cytoplasmic
  • All cells contain
    • Same core structure
    • Same motors Dynein (-) and Kinesin (+)
    • Different associated proteins
  • Dynamic
    • Continuous remodelling
  • Movement
    • Intracellular > cellular
    • Cell division mitotic spindle
  • Specialized structures
    • centrosome, basal bodies, Spindle pole
    • Cell processes – cilia (9+2)

Prokaryotic Cytoskeleton Filaments

Prokaryotic cells have recently been shown to contain a number of proteins that appear to be analogous to eukaryotic cell cytoskeletal structures and functions.

FtsZ ring

  • microtubule homolog
  • dynamic and exchanges subunits with the cytoplasmic pool
  • assembles into a ring at the future site of bacterial septum in cell division

MreB

  • microfilament (actin) homolog
  • dynamic and exchanges subunits with the cytoplasmic pool
  • assembles into helix-like structures
  • thought to spatially restrict cell growth activities during cell elongation

Crescentin

  • intermediate filament homolog
  • form stable filamentous structures
  • continuously incorporate subunits along their length
  • grow in a nonpolar fashion
  • stably anchored to the cell envelope

Links: Nature Cytoskeleton Milestones 1992–1998 Discovery of the bacterial cytoskeleton

Intermediate Filaments

Unlike the microfilament and microtubule systems, the filaments themselves consist of a wide variety of different proteins. These intermediate filaments have important structural roles in cell integrity both internally and through specialized cellular junctions that occur between cell-cell and cell-matrix which surrounds them. This topic will be addressed again when we look at the cell cytoskeleton and the extracellular matrix.

Physical Characteristics

  • 10 nm diameter
  • Named by size relative to other cytoskeletal filaments
  • intermediate filaments have no structural polarity
  • Monomer – central α-helical domain
  • Dimer – 2 monomers form parallel coiled coil
  • Tetramer – pair of parallel dimers associates in an antiparallel staggered fashion
    • tetramer is the soluble subunit (analogous to MT αβ-tubulin dimer, or MF actin monomer)
  • Provide rope-like resistance to mechanical stress
  • In muscle- link Z discs of adjacent myofibrils
  • Organization can be altered by phosphorylation

IF Types

  • Type I (n = 28)
    • Acidic keratins (pI < 5.7) 40–64 kDa
      • K9-28 (epithelia)
      • K31-40 (hair/nail)
  • Type II (n = 26)
    • Basic keratins (pI ≥ 6.0) 53–67 kDa
      • K1-8, K71-80 (epithelia)
      • K81-86 (hair/nail)

Keratins form heterodimers that assemble into heteropolymeric keratin filaments

  • Type III
    • Desmin (cardiac, skeletal and smooth muscle)
    • Vimentin (widespread distribution: leukocytes, blood vessels, endothelial, some epithelial and mesenchymal cells) 56 kDa
    • Peripherin (neurons) 57 kDa
    • Glial fibrillary acidic protein (GFAP) (astrocytes/glia) 50 kDa

Type III intermediate filament proteins can form both homo- and heteropolymeric filaments

  • Type IV
    • Neurofilament Low NF-L (neurons) 62 kDa
    • Neurofilament Medium NF-M (neurons) 110 kDa
    • Neurofilament High NF-H (neurons) 130 kDa

Neurofilaments form heteropolymers

  • α-internexin (CNS neurons)
  • Synemins (muscle)
  • Syncoilin (muscle)
  • Nestin (stem cell marker) 220 kDa
  • Type V
    • Lamin A/C (ubiquitous) 62–72 kDa
    • Lamin B1/2 (ubiquitous) 65–68 kDa
  • Orphan
    • Phakinin (lens)
    • Filensin (lens)

Intermediate Filament Associated Protein (IFAP)

  • Cross-link intermediate filaments with one another
    • forming a bundle (also called a tonofilament) or a network
  • IFAPs
    • Plectin 500 kDa Striated muscle, epithelia Nuclear envelop
    • Syncoilin 64 kDa Striated muscle
    • Nesprin-3 117 kDa Kerotinocytes
    • Paranemin 280 kDa
    • Desmuslin 230 kDa

Function

  • found in some metazoans (vertebrates, nematodes, and molluscs)
  • Desmin interacts with nebulin linking intermediate filament network and sarcomeres at the Z-discs
  • Keratin filament formation originates mainly from sites close to the actin-rich cell cortex
  • 2 alternate theories as to IF load/strain transmission
    • entropic gels – where no individual intermediate filaments experiences direct loading in tension
    • single intermediate filaments and bundles – extensible and elastic in vitro, and therefore well-suited to bearing tensional loads

Applications

Take advantage of the unique cell type expression pattern of IF proteins.

  • Nestin as a stem cell marker
  • GFAP can be used as an astrocyte marker in the analysis of neuronal tissue
  • Vimentin antibody can be used as a neural stem cell marker (ab45939)
  • Vimentin is highly expressed in fibroblasts and some expression in T- and B-lymphocytes. Expressed in many hormone-independent mammary carcinoma cell lines.

Microtubules

Microtubules are the largest filament system of the cytoskeleton and have important functions for intracellular motility of nearly all cytoplasmic structures (organelles, vesicles, and smaller components).

(MH – Note that the role of microtubules in mitosis will not be covered in detail, as this topic is covered elsewhere in lecture series)

The key concepts are: microtubules, intracellular motility, tubulin, microtubule associated proteins, microtubule motors, centrosome, flagella, cilia The lecture slides and textbook alone contain enough information as an introduction to the subject for this level of study. If you are interested in further reading, I have also included below links to more detailed textbooks with further information and images. Please note this additional information is not necessarily examinable, but may be useful if you have not previously studied biology.

About Microtubules

  • Cell organizing role
  • Cytoskeleton
    • Largest fibre
    • 25 nm diameter
    • cytoplasmic
  • All cells contain
    • Same core structure
    • Same motors
    • Different associated proteins
  • Dynamic
    • Continuous remodelling
  • Movement
    • Intracellular > cellular
    • Cell division

Motility- Intracellular

organelle movement vesicle transport mitosis & meiosis chromosome segregation gene expression transcription factor binding mRNA transport translation protein export transmitter release Motility- Intracellular Axon transported vesicles EM axonal transported vesicles and axonal cytoskeleton in longitudinal section Arrows show rod shaped structures appear as cross bridges between organelles and microtubules Scale bar 100 nm

Basal Bodies of Cilia

Surface of ependymal cell contains basal bodies red rings connected to cilia microtubules longitudinal section Inset: cilia transverse section central MT doublet surrounded by nine MT pairs one of each pair has a hook-like appendage (arrows) Å~100,000 see later motor slides

Endocytic Pathway

Endocytic movement occurs along microtubules can be blocked by drugs Depolymerizing drugs Stabilizing drugs

Structure

Long hollow tubes 25 nm diameter Radiate from forming structure Centrosome Spindle pole Basal Body Polarized (+) plus and (-) minus ends Formed from Tubulin 55 kD protein

Microtubule Structure

(A) EM of mt in cross-section ring of 13 distinct subunits Each a separate tubulin molecule an alpha/beta heterodimer (B) EM of a mt assembled in vitro (C) 13 molecules in cross-section (D) side view of a mt tubulin molecules aligned into long parallel rows 13 Protofilaments Each is composed of a series of tubulin molecules, each an a/b heterodimer mt is a polar structure with a different end of tubulin molecule (a or b) facing each end of microtubule

Tubulin Protofilaments

dimers polymerize to form microtubules 13 linear protofilaments head-to-tail arrays of tubulin dimers arranged in parallel assembled around hollow core

Microtubule Polarity

Tubulin subunits in a MT subunits aligned end to end into a protofilament magenta highlight side-by-side protofilament packing forms wall of microtubule slightly staggered so that a-tubulin in one protofilament contacts b-tubulin in neighboring protofilaments

Arrangement of Protofilaments

Singlet typical microtubule tube built from 13 protofilaments Doublet additional set of 10 protofilaments form a second tubule by fusing to the wall of a singlet Triplet Attachment of another 10 protofilaments

Tubulin

  • dimer 55-kd polypeptides
    • α-tubulin (alpha-)
    • β-tubulin (beta-)
  • encoded by related genes
  • third type of tubulin
    • γ-tubulin (gamma-)
  • located at centrosome
  • role in initiating mt assembly

Tubulin Genes

  • human DNA contains about 14 copies per genome of both genes Cleveland et al. (1980)
    • Beta β 6p21.3 – 15 to 20 members
    • Alpha α mainly Chr.12 – 15 to 20 dispersed genes
    • Gamma γ 17q21
  • Also many tubulin pseudogenes

Tubulin Synthesis Regulation

autoregulation in animal cells stability of polysome-bound tubulin mRNAs beta-tubulin RNAs selectively targeted as substrates for destabilization not recognition of specific RNA sequences co-translational recognition of amino-terminal beta-tubulin tetrapeptide after emergence from ribosome Motif could be used in other systems where RNA degradation is coupled to ribosome attachment and translation

Tubulin Homology

FtsZ bacterial GTPase (40,000 Mr) bacterial protein has structural and functional similarities with tubulin ability to polymerize and a role in cell division protein carrying out these ancestral functions in bacteria was modified during evolution to fulfill diverse roles of microtubules in eukaryotes?

Centrosome

slow-growing minus end of MT embedded in centrosome matrix surrounding a pair of centrioles matrix determines number of MTs in a cell By nucleating growth of new MTs Microtubule Organization Movie: Microtubules and Mitochondria Movie: Microtubules and Endoplasmic Reticulum

Centrosome Cycle

Orientation of MTs in Cells (-) Minus ends of MTs generally embedded in a microtubule-organizing center (mtoc) alpha (+) plus ends often located near the plasma membrane beta

MT Treadmilling

Treadmilling dynamic behavior when tubulin bound to GDP continually lost from minus end replaced by the addition of tubulin bound to GTP to plus end of same microtubule GTP hydrolysis also results in dynamic instability individual microtubules alternate between cycles of growth and shrinkage

Microtubule Movement

GTP hydrolysis destabilizes MTs Addition of tubulin adds GTP to end of protofilament grows in linear conformation readily packed into MT wall becoming stabilized Hydrolysis of GTP changes subunits conformation force protofilament a curved shape less able to pack into the MT wall protofilaments with GDP-containing subunits forced linear conformation by lateral bonds within MT wall, mainly in stable cap of GTP-containing subunits

GTP Cap Hydrolysis

GTP hydrolysis destabilizes MTs GDP-containing protofilaments relax curved conformation progressive disruption of MT disassembly of protofilaments free tubulin dimers

Microtubule Associated Protein

  • MAP2
  • Neuron expression
  • a 280-kD protein
  • concentrated in neuronal soma and dendrites
  • Developmentally regulated expression
  • 2q34-q35
  • MAP2 Developmental Expression
    • MAP2B – present throughout brain development
    • MAP2A – appears during end of second week of postnatal life
    • MAP2C – present during early brain development, disappears from the mature brain, except for the retina, olfactory bulb, and cerebellum
  • MAP2A and MAP2B
    • encoded by 9-kb mRNAs
  • MAP2C
    • encoded by a 6-kb mRNA

Tau

  • Protein Mr 45-60 kDa
  • Gene 17q21.1

Neuron Expression Enriched in axons phosphorylated Tau- Alzheimer Disease neuronal cytoskeleton is progressively disrupted replaced by tangles of paired helical filaments (PHFs) PHFs composed mainly of hyperphosphorylated form of Tau Tau- Alzheimer Disease Elevated tau inhibit intracellular transport mainly plus-directed transport (kinesin motors) from center of cell body to neuronal processes organelles are unable to penetrate neurites peroxisomes, mitochondria, and transport vesicles carrying supplies for growth cone Leads to stunted growth increased susceptibility to oxidative stress pathologic aggregation of proteins such as amyloid precursor protein (APP) tau:tubulin ratio is normally low increased levels of tau become detrimental to the cell

Microtubule Motors

Microtubule Motor Proteins

  • Dynein (-) and Kinesin (+) move in opposite directions
  • globular heads of heavy chains bind mts
  • motor domains

Dynein

  • – (minus) end motor
  • 2 or 3 heavy chains (two are shown here)
  • multiple light and intermediate chains

Kinesin

  • + (plus) end motor
  • 2 heavy chains, wound around each other in a coiled-coil structure
  • 2 light chains
  • Movie: Kinesin on Microtubule
  • Movie: Microtubules in vitro

Ciliary and Flagellar Axonemes

  • 9 + 2 MT arrangement
  • dynein arms and radial spokes with attached heads occur at intervals along the longitudinal axis
  • central microtubules, C1 and C2
  • Axonemal Dynein
    • Arrangement of globular domains and short stalks
    • attachment of outer dynein arm to the A tubule of one doublet and cross-bridges to B tubule of an adjacent doublet
    • attachment to A tubule is stable

Presence of ATP successive formation and breakage of cross-bridges to adjacent B tubule leads to movement of one doublet relative to the other Dynein-mediated sliding of axonemal mt Dynein arm attach to A subfiber of one microtubule walk along B subfiber of adjacent doublet toward (-) end (small arrow) moves microtubule in opposite direction (large arrow) cross-links (nexin) broken, sliding can continue

Microtubules In Development

  • nurse cells in insect ovarioles
  • supply oocytes cellular components
  • mRNAs, proteins
  • pass from one cell to another through intercellular bridges traversed by microtubules
  • mRNAs encode axis-determining factors in Drosophila embryos
  • mRNAs are translocated
  • localized within oocyte
  • sites where translation products will function

Microtubule Drugs and Cancer

  • drugs affect microtubule assembly
  • experimental cell biology tool
  • Cancer treatment
  • Colchicine and Colcemid
    • bind tubulin
    • inhibit mt polymerization, blocks mitosis
  • Vincristine and Vinblastine
    • cancer chemotherapy
    • selectively inhibit rapidly dividing cells
  • Taxol
    • stabilizes microtubules rather than inhibiting their assembly
    • also blocks cell division

Taxol – Paclitaxel

  • 1971 from the bark of the Western yew
  • Taxus brevifolia Nut (Taxaceae)
  • Anti-tumor and anti-leukemic activity
  • found in roots, leaves, and stems of this and related members of yew family
  • complex ester
  • an oxetan ring attached to a derivative of taxane
  • tool for investigating MT function
  • clinical trials in a variety of cancers
  • Initial development limited by low abundance in yew trees
  • now novel synthetic methods
  • identification of new sources of taxanes

Cytoskeleton – Microfilaments

This lecture introduces the smallest of the three cytoskeleton filament systems, microfilaments.

  • Microfilaments are made up of actin subunits (G-actin monomers)
  • G-actin (globular actin) monomers are globular proteins of 375 amino acids
  • Each G-actin monomer is described as having both a barbed end, and a pointed end
  • Microfilaments have an approximate diameter of 8nm
  • G-actin monomers polymerise to form actin filaments (F-actin)
  • F-actin threads associate with each other in a thin double-helical structure

Actin filaments are thin, relatively flexible threads that can be crosslinked together in different ways to form very different structures. Depending on the cell type and activity, an actin filament can have a different structural and dynamic properties.

FUNCTION

  • CELL MOTILITY
    • Pseudopodia
      • Filopodia and lamellipodium
    • Actin microfilaments are widely essential for cell motility, and they are therefore widely distributed in the very mobile cells in the body
  • INTRACELLULAR TRANSPORT
    • Actin microfilaments play in key role in the ability of cells to transport their intracellular organelles between cells during cell division.
    • In addition, actin filaments form a track system allowing cargo transport. Actin microfilaments interact with proteins of the myosin family in order to move vesicles and organelles within cells.
  • CONTRACTION OF MUSCLE CELLS:
    • In muscle cells, the thin filaments (actin) and the thick filaments (myosin), arrange into actomyosin myofibrils which are responsible for mediating muscle cell contraction. The sliding past of actin filaments is the key principle behind the muscle contraction mechanism. Contraction is achieved by the movement of myosin which attached to the actin microfilaments in a process called cross bridge cycle. Myosin heads use the energy from ATP hydrolysis to exert a tension that which creates a power stroke, causing the sliding of actin filaments and shortening the muscle
  • DETERMINING CELL SHAPE
    • Actin filaments are situated in high concentrations at the periphery of the cell, forming a three dimensional network beneath the plasma membrane. This network of actin filaments with the association with actin-binding proteins together are called the cell cortex, which determines the cell shape, as well as a variety of cell surface activities, like movement.

Chapter 20 Cellular Communities:Tissues, Stem Cells, and Cancer – Extracellular Matrix and Connective Tissues pp 690-699


Introduction

This lecture introduces the materials lying outside the cell, known collectively as the extracellular matrix (ECM). There is no one matrix though, with different tissues having their own specific ECM, which may be dynamic or static in structure. In particular the ECM has significant roles in normal tissue development, function and disease. This matrix is manufactured by cells, secreted and modified outside the cell by several different enzymes.

ECM Function

  • Support for cells
  • Pattern of ECM regulates
    • polarity
    • cell division
    • adhesion
    • motility
  • Development
    • migration
    • differentiation
    • growth factors

ECM Features

  • stable and able to be reorganised?
  • different for different tissues

ECM Structure

  • Glycoproteins
  • Fibers
    • Collagen- main fibers
    • Elastin
  • Hydrated Matrix
    • Proteoglycans
    • high carbohydrate
  • Adhesive
    • Laminin
    • Fibronectin

Shapes and Sizes ECM molecules

Collagen

  • tensile strength and elasticity
    • Tendons
    • Cartilage
    • Bone
  • half total body proteins (by weight)

Collagen Components

  • Insoluble glycoprotein
    • protein + carbohydrate

Protein

  • high glycine
  • high proline
  • hydroxylysine
  • hydroxyproline
  • (gly-X-Y)n

Carbohydrate

  • glucose
  • galactose

Collagen Structure

Collagen Protein

  • 3 polypeptide (a) chains
  • left hand helix, forms fibers
  • many different (vertebrate) collagens by different combinations of a-chains
  • Type I, II, III
    • main fibers, flexible
  • Type I
    • bone, skin, tendons
    • 90% of all collagen
  • Type II
  • cartilage

Collagen Fibers

  • Type I, II, III cross striated
    • e.g. tendons – type I fibrils, have a 67-nm period striations and are oriented longitudinally (direction of the stress)
    • showing overlapping packing of individual collagen molecules
    • reticular fibres type III, support individual cells
  • Type IV fine unstriated
    • sheet-like supportive meshwork
    • mature basal laminae
    • tracks for embryonic migration
    • barriers for cell migration
  • Type V-XII
    • smaller diameter fibers than I-III
    • no striations

Collagen Interactions

Collagen fibril types can interact with a variety of non-fibrous collagen types (microfiber)

  • fibrous collagens—types I, II, III, and V
  • Cartilage – types II (fiber) and IX collagen microfibrils
  • Tendons – type I fibrils bound and linked by type VI microfibrils.

Collagen Type Functions

  • Collagen Type I – skin, tendon, vascular, ligature, organs, bone (main component of bone)
  • Collagen Type II – cartilage (main component of cartilage)
  • Collagen Type III – reticular fibers with type I.
  • Collagen Type IV – forms bases of cell basement membrane

Collagen Synthesis

Endoplasmic ReticulummRNA attached to ERprotein synthesized into ER lumencotranslational and post-translational modifications3 proto-a-chains form soluble procollagenmoved to golgi apparatusGolgi Apparatuspacked into secretion vesiclesfuse with membraneOutside Cellprocollagen processed by enzymes outside cellassemble into collagen fiberscollagen fibrils form lateral Interactions of triple helices
Collagen DiseasesCollagen Diseases – Excessfibrosislung- pulmonary fibrosisoverproduction of collagen Iliver- over consumption of alcoholarteries- atherosclerosis

Collagen Diseases – Insufficient

  • Ehlers-Danlos syndrome
    • rubber-man
    • skin and tendons easily stretched
    • contortionists often suffer from this disease
  • Osteogenesis imperfecta
    • brittle-bone syndrome
    • mutation in Type I procollagen
    • fail to assemble triple helix
    • degrade imperfect collagen
    • Leads to fragile bones
  • Scurvy
    • dietary Vitamin C deficiency
    • needed for hydroxylation
    • Proline -> Hydroxyproline
    • form too few hydrogen bonds in collagen
    • skin, bone, teeth weakness and malformation
    • blood vessels weakened, bleeding

Elastin

  • elastin and elastic fibres
    • uncoils into an extended conformation when the fiber is stretched
    • recoils spontaneously as soon as the stretching force is relaxed

Elastic fibers are composed of a core of cross-linked elastin embedded within a peripheral mantle of microfibrils.

Microfibrils

  • may regulate assembly and organization of elastic fibers by acting as a scaffold
  • guiding tropoelastin deposition
  • aggregates of threadlike filaments
  • periodically spaced globular domains (beads) connected by multiple linear arms
    • beaded structure is parallel fibrillin monomers aligned head-to-tail
  • fibulin-5 induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils

Elastin Structure

  • composed of the amino acids glycine, valine, alanine, and proline
  • cross-linked tropoelastin monomers
  • first secreted as soluble precursors (tropoelastin)
  • assembly and crosslinking of tropoelastin monomers
  • form insoluble elastin matrix into functional fibres
    • lysine residues in the cross-linking domain of secreted tropoelastin rapidly cross-linked (both inter- and intra-molecularly by lysyl oxidase)
    • hydrophobic segments – elastic properties
    • α-helical segments (alanine- and lysine-rich) – form cross-links between adjacent molecules

Elastin Function

  • structural integrity and function of tissues
  • requiring reversible extensibility or deformability
  • high levels in tissues that require elasticity
    • lung, skin, major blood vessels

Proteoglycans

  • consist of protein (~5%) and polysaccharide chain (~95%)
  • form a gel to embed the fibril network
  • Golgi apparatus – GAG disaccharides are added to protein cores to form proteoglycans
  • 10% by weight but fill most of space
  • unbranched polysaccharide chains
  • disaccharide subunits
  • amino sugar

Glycosaminoglycans (Gags)

  • Hyaluronan (or hyaluronic acid) main glycosaminoglycan in connective tissue
  • high molecular weight (~ MW 1,000,000 )
  • length of about 2.5 µm hyaluronan
  • “backbone” for the assembly of other glycosaminoglycans
    • Hyaluronan is also a major component of the synovial fluid, which fills joint cavities, and the vitreous body of the eye.
  • Other 4 major glycosaminoglycans
    • chondroitin sulphate, dermatan sulphate, keratan sulphate and heparan sulphate (UK sulphate, US sulfate)
    • attach through core and link proteins to hyaluronic acid backbone

Proteoglycan Function

  • trap water
  • resistant to compression
  • return to original shape
  • occupy space
  • link to collagen fibers
  • form network
    • in bone combined with calcium hydroxyapatite, calcium carbonate

Development

  • produce a “cell-free” space
  • for cell proliferation and migration into
  • heart, cornea

Adult

  • in areas of compression
  • tissues, joints

ECM Function

  • Support for cells
  • Pattern of ECM regulates
    • polarity
    • cell division
    • adhesion
    • motility
  • Development
    • migration
    • differentiation
    • growth factors

ECM stable and able to be reorganised?

ECM Structure

  • Glycoproteins
  • Fibers
    • Collagen- main fibers
    • Elastin
  • Hydrated Matrix
    • Proteoglycans
    • high carbohydrate
  • Adhesive
    • Fibronectin
    • Laminin

Cell Adhesion to ECM

  • Direct linkage to collagen or proteoglycan
  • insertion of fibers into membrane
  • covalent attachment to membrane lipid
  • Linking glycoproteins
    • fibronectin
    • laminin

Fibronectin

  • dimer connected at C-terminal
    • Mr 550 kDa
    • nearly identical subunits composed of types I (F1), II (F2), and III (F3) fibronectin modules
  • S-S linkages
  • rigid and flexible domains
  • fibronectin fibrils have elastic properties and can stretch fibrils up to four-fold their relaxed length.
  • fibrillogenesis – transformation from the compact (soluble) form to the extended fibrillar (insoluble) form of fibronectin, requires application of mechanical forces generated by cells.

Fibronectin Function

  • soluble protein in blood plasma (200–250 kDa monomer)
    • blood clotting process, link to fibrin
  • insoluble protein in extracellular matrix (ECM)
    • ECM fibronectin differs from plasma fibronectin by the presence of additional polypeptide segments and in altering morphology of transformed cells and hemagglutination.

Laminin

  • cross-shaped glycoprotein
  • 3 polypeptides a, b1, b2
  • carbohydrate (13% by weight)
  • Mr 900K
  • separate binding domains
    • collagen IV
    • heparin
    • heparin sulphate
    • cell binding
    • cell specific binding – liver, nerve
    • cell surface receptor

Laminin Function

  • cell adhesion
  • migration pathways
  • stimulates growth of axons
  • development and regeneration
  • differentiation
  • basal laminae
  • most abundant linking glycoprotein

Basement membrane

The epithelial ECM the term “basement membrane” is used with light microscopic observation and “basal lamina” is used with electron microscopy.

The basement membrane is composed of two sublayers.

  1. Basal lamina
  • (about 40–120 nm thick) consists of fine protein filaments embedded in an amorphous matrix.
  • Membrane proteins of the epithelial cells are anchored in the basal lamina, which is also produced by the epithelial cells.
  • major component of the basal lamina are two glycoproteins – laminin and (usually type IV) collagen

2)   Reticular lamina

  • consists of reticular fibres embedded in ground substance.
  • fibres of the reticular lamina connects the basal lamina with the underlying connective tissue.
  • components of the reticular lamina are synthesised by cells of the connective tissue underlying the epithelium.

Chapter 18 The Cell Division Cycle – Overview of the Cell Cycle pp 609-624


Prokaryote Division

  • Binary Fission – and seen in eukaryote mitochondria
  • Asexual reproduction – replicates original cell to produce two identical cells
  • Grow in numbers exponentially – adequate nutrients and a fast life cycle
  • single organism can multiply into billions
  • High mutation rate of bacteria

Appear to involve proteins that are homologs of eukaryotic cytoskeleton proteins. Prokaryotic Cytoskeleton Filaments

Cell Lifespan

  • Body Cell Types – about 210 types
  • Lifespan
    • Born
    • Differentiate
    • Function
    • Die or Divide

Cell Lifespan Examples

Neutrophil 6-7 hours circulating 4 days in tissue Red blood cell 120 days Brain neuron, heart 50 – 100 years

Lifespan Processes

  • Birth – Mitosis (except germ cells – Meiosis)
  • Growth – Expression of genes and proteins required to grow the cell, its organelles and cytoskeleton
  • Function – Expression of tissue specific genes and proteins
  • Division – DNA during cycle, whole cell in Mitosis
  • Death – Apoptosis (programmed cell death) Necrosis (un-programmed cell death)

Cell Cycle Major Phases

  • Mitosis (M phase)

Cell birth(division) small time of cell cycle

  • Interphase

Most cell life Cell growth, function DNA synthesis organelle development

Cell Cycle- Stages

Rapidly dividing cell (20-24hr)

Mitosis

  • M phase 1 hr

Interphase

  • G1 Phase
    • cellular growth 9hr
    • Most variable time
    • Can exit to G0
  • S Phase
    • DNA duplication 9hr
  • G2 Phase
    • growth prepare for mitosis 4 hr

Cell Cycle Differences

Early Embryonic Cycle

  • no growth occurs
  • each daughter cell is half the size of parent cell
  • cycle time is very short
  • S phases and M phases alternate without any intervening G1 or G2 phases

G0 Phase

  • exits the cycle at G1 (cancer cells do not enter G0)
  • cell can leave the cell cycle (temporarily or permanently)
  • temporarily – quiescent
  • permanently – terminally differentiated
    • cell will never reenter the cell cycle
    • carry out their function until they die
  • not simply the absence of signals for mitosis
  • active repression of the genes needed for mitosis

Cell Cycle Regulation

  • Cell proliferation is strictly regulated
  • Unregulated/abnormal proliferation is oncogenesis or Cancer

Cell Cycle- External Regulators

  • Cell replacement in different tissues
    • regulated by growth factors
    • can be specific for specific cell types

Growth Factors and Cell Cycle Progress

  • External factors can also regulate progression through cycle
  • Growth factors primarily act on cells in G0 and G1
  • The restriction point is the timepoint in G1 when cells no longer respond to withdrawal of growth factors by returning to G0, but progress to S phase.
    • thought to involve retinoblastoma protein (pRb)

Growth Factor Model

  • Fibroblasts in culture
    • Serum (Prepared by clotting)- Proliferation
    • Plasma (Prepared by centrifugation, no clotting)- no proliferation
  • Clotting
    • allows platelets to release secretory granules
    • Platelet-derived growth factor (PDGF)
  • Connective tissue cells express PDGF receptors which bind the small PDGF glycoprotein

Other Growth Factors

  • Interleukin-2 (IL-2)
    • Stimulates T lymphocytes
  • Nerve Growth Factor (NGF)
    • Promotes neuronal survival and growth
  • Epidermal Growth Factor (EGF)
  • Vascular Endothelial Growth Factor (VEGF)
  • Insulin-like growth factors (IFG-1, IGF-2)

Cell Cycle- Internal Regulators

1980s – studies in Xenopus eggs and starfish oocytes purified M phase-promoting factor (MPF) from and the identification of its components as cyclin B and CDC2 (also called cyclin-dependent kinase

Cyclins

Cyclins are synthesized and degraded each cell cycle (hence the name)

Cyclins and Cyclin-Dependent Kinases need to interact for cell cycle progression Cyclins and Cyclin-Dependent Kinases

Cyclin D

  • cyclin D1, D2 and D3
  • expression induced by growth factors stimulation
    • serum growth factors to quiescent cells promotes transcription of the cyclin D1 gene
  • Cyclin D1 then binds Cdk4 and Cdk6 in early to mid-G1 phase
    • phosphorylate and inactivate retinoblastoma protein (pRb)
  • also acts as a cofactor for several transcription factors in numerous cell types

Cyclin D1 is a proto-oncogene

  • mutations are associated with cancer progression
  • as a regulator of G1 to S-phase transition

Cyclin E

  • required for transition from G1 to S phase
  • Cyclin E binds to G1 phase Cdk2
  • Cyclin E/Cdk2 complex phosphorylates p27Kip1 which then degrades
    • an inhibitor of Cyclin D
  • activation of Cyclin E gene can be blocked by the cdk inhibitor p16 (Cyclin-dependent kinase inhibitor 2A)
    • tumor suppressor protein
  • expression of Cyclin A then increases for progress to S phase

Cyclin A

  • accumulates from early S phase
    • role not fully understood, required for S phase progress
  • Cyclin A binds Cdk2
  • disappears ahead of cyclin B during mitosis
  • can bind to both Cdc2 and Cdk2

Cyclin B

  • accumulates from S phase
  • Cyclin B forms a complex with Cdc2
    • complex is kept inactive by phosphorylation of Cdc2
    • abruptly activated by Cdc25 during mitosis
  • cyclin B is destroyed at mitosis exit by ubiquitin-mediated mechanism (catalyzed by the APC/C)

Anaphase-Promoting Complex (APC, cyclosome, APC/C)

  • degrades the mitotic (B) cyclins
  • triggers the events leading to destruction of the cohesins
  • allowing the sister chromatids to separate

Cyclin-dependent Kinases

  • Inactive until bound to a specific cyclin
  • kinase – means an enzyme which phosphorylates a target protein(s)
  • Drive M to S Phase
    • cdk1 and cdk2
  • Cdk1 activated at G2 to M
  • Cdk2 activated at G1 to S

Cell Cyclin Changes

Interphase and M Phase

  • Division controlled by synthesis/degradation cyclin B
  • regulatory subunit of Cdc2 protein kinase
  • interphase cyclin B synthesis leads to formation of active cyclin B–Cdc2 complex
  • induces entry into mitosis

Rapid degradation of cyclin B leads to inactivation Cdc2 kinase

  • Allow cell to exit mitosis and return to interphase next cell cycle
  • cyclin B-CDC2 acts as M phase-promoting factor (MPF)
    • activate other proteins through phosphorylation
  • cyclin A down-regulation induces a G2 phase arrest through a checkpoint-independent inactivation of cyclin B-CDC2 by inhibitory phosphorylation.
    • cyclin A cannot form MPF independent of cyclin B

Regulator Checkpoints

These regulators are often described as “tumor suppressor proteins” due to their ability to block tumor (cancer) growth. Conversely, mutations in these genes often lead to tumor growth.

Checkpoints in the cell-cycle control system

How DNA damage arrests the cell cycle in G1

p53

  • (TP53) A multifunctional protein Mr 53 kDa regulating cell cycle and apoptosis
  • As a cell cycle regulator it recognizes and binds damaged DNA
    • single-stranded DNA, insertion/deletion mismatches, and free DNA ends
  • Acts as a transcription factor activating p21 transcription
    • p21 protein then inhibits G1 cyclin-dependent kinases p21

pRb

  • retinoblastoma protein regulating cell cycle
  • As a cell cycle regulator it recognizes damaged DNA
  • binds and inhibits transcription factors of the E2F family
    • active pRb is hypophosphorylated
    • inactive pRb is phosphorylated
  • mutations in this gene lead to the disease retinoblastoma OMIM 180200

p16

  • also called Cyclin-dependent kinase inhibitor 2A
  • blocks activation of Cyclin E gene
  • mutations found with
    • pancreatic adenocarcinoma OMIM 260350
    • esophageal and gastric cancer cell lines

Binary Fission

  • Prokaryotes – And Eukaryote mitochondria
  • Asexual reproduction – replicates original cell to produce two identical cells
  • Grow in numbers exponentially
    • adequate nutrients and a fast life cycle
    • single organism can multiply into billions
  • High mutation rate of bacteria

(fission = splitting of something into its parts)

Cell Lifespan

  • Body cell types – About 210 types
  • Lifespan – Born, Differentiate, Function, Divide or Die

Cell Types

  • Neutrophil – 6-7 hours circulating – 4 days in tissue
  • Red blood cell – 120 days
  • Brain neuron, heart – 50 – 100 years

Cell Changes

  • Nucleus
    • Chromosome condensation
    • Nuclear envelope breakdown
  • Cytoplasm
    • Cytoskeleton reorganization
    • Spindle formation (MT) Contractile ring (MF)
    • Organelle redistribution
  • Mitosis Energy
    • Cell division uses up a lot of energy, so cells ensure they have enough resources to complete the job before committing to it.

Mitosis Phases

  • Based on light microscopy of living cells light and electron microscopy of fixed and stained cells
  • 5 Phases – prophase, prometaphase, metaphase, anaphase, and telophase
    • Cytokinesis 6th stage overlaps the end of mitosis

Interphase

  • not a mitotic phase (discussed in cell cycle)
  • Chromosomes dispersed in nucleus
  • Gene expression
  • Cytoskeleton and cell organelles – Distributed and functioning
  • Mitochondria undergo independent proliferation/division

Chromosome Changes

Prophase

  • Chromosome DNA has been earlier duplicated (S Phase)
  • Chromosomes begin condensing
  • Chromosome pairs (chromatids) held together at centromere
  • Microtubules disassemble
  • Mitotic spindle begins to form

Spindle Apparatus

  • 3 sets of microtubules – (+) ends point away from centrosome at each pole.
  1. astral microtubules – anchor the pole end in position
  2. kinetochore microtubules – connected to chromosomes
  3. polar microtubules – form the structure of the spindle apparatus

At end of prophase nuclear envelope breaks down

Prometaphase

  • Microtubules now enter nuclear region
  • Nuclear envelope forms vesicles around mitotic spindle
  • Kinetochores form on centromere attach to some MTs of spindle

At end of prometaphase chromosomes move to metaphase plate

Metaphase

  • Kinetochore MTs align chromosomes in one midpoint plane

Metaphase ends when sister kinetochores separate

Anaphase

  • Separation of sister Kinetochores
  • shortening of Kinetochore microtubules pull chromosomes to spindle pole

Anaphase ends as nuclear envelope (membrane) begins to reform

Telophase

  • Chromosomes arrive at spindle poles
  • Kinetochore MTs lost
  • Condensed chromosomes begin expanding
    • Continues through cytokinesis

Cytokinesis

  • Division of cytoplasmic contents
  • Contractile ring forms at midpoint under membrane
  • Microfilament ring – contracts forming cleavage furrow
    • myosin II is the motor
  • Eventually fully divides cytoplasm

Cell Organelles

Mitochondria

  • Divide independently of cell mitosis
  • distributed into daughter cells

Peroxisomes

  • localise at spindle poles

Endoplasmic Reticulum

Golgi

  • 2 processes – disassembly and reassembly
  • Golgi stack undergoes a continuous fragmentation process
  • fragments are distributed into daughter cells
  • are reassembled into new Golgi stacks

Disassembly

  • Unstacking – mediated by two mitotic kinases (cdc2 and plk)
  • Vesiculation – mediated by COPI budding machinery ARF1 and the coatomer complex

Reassembly

  • Fusion – formation of single cisternae by membrane fusion
  • Restacking – requires dephosphorylation of Golgi stacking proteins by protein phosphatase PP2A

Mitosis and Meiosis

Progeny

Mitosis 2 Daughter cells identical to parent (diploid)

Meiosis Germ cell division (haploid)

  • Reductive division
  • Generates haploid gametes (egg, sperm)
  • Each genetically distinct from parent
  • Genetic recombination (prophase 1)
    • Exchanges portions of chromosomes maternal/paternal homologous pairs
  • Independent assortment of paternal chromosomes (meiosis 1)

Cell Birth – Mitosis and Meiosis 1st cell division- Meiosis

Homologous chromosomes pairing unique to meiosis

  • Each chromosome duplicated and exists as attached sister chromatids before pairing occurs
  • Genetic Recombination shown by chromosomes part red and part black
    • chromosome pairing in meiosis involves crossing-over between homologous chromosomes

(For clarity only 1 pair of homologous chromosomes shown)

Comparison of Meiosis/Mitosis

  • After DNA replication 2 nuclear (and cell) divisions required to produce haploid gametes
  • Each diploid cell in meiosis produces 4 haploid cells (sperm) 1 haploid cell (egg)
  • Each diploid cell mitosis produces 2 diploid cells

Abnormalities

Meiotic Nondisjunction

  • Occurs when homologues fail to separate during meiotic division I or II
  • Down Syndrome
  • Caused by an extra copy of chromosome 21

Chromosomal Translocations

  • Philadelphia chromosome
  • Chronic myelogenous leukemia
    • Piece of Chr9 exchanged with Chr22 Generates truncated abl

Overstimulates cell production

Meiosis Sex Differences

Female (oogenesis)

  • Meiosis initiated once in a finite population of cells
  • 1 gamete produced / meiosis
  • Completion of meiosis delayed for months or years
  • Meiosis arrested at 1st meiotic prophase and reinitiated in a smaller population of cells
  • Differentiation of gamete occurs while diploid in first meiotic prophase
  • All chromosomes exhibit equivalent transcription and recombination during meiotic prophase

Male (spermatogenesis)

  • Meiosis initiated continuously in a mitotically dividing stem cell population
  • 4 gametes produced / meiosis
  • Meiosis completed in days or weeks
  • Meiosis and differentiation proceed continuously without cell cycle arrest
  • Differentiation of gamete occurs while haploid after meiosis ends

Sex chromosomes excluded from recombination and transcription during first meiotic prophase

Cell Death

Cell Recycling

There are a number of different cellular mechanisms and processes for reusing cellular components or removing abnormal products. These pathways can also be utilized during periods when the cell is placed under specific or limited growth conditions. Autophagy is an important part of this process, but will be covered under the Cell Death section of this current lecture.

Proteasome

Figure 6-88. Two general ways of inducing the degradation of a specific protein Figure 6-86. The proteasome

Ubiquitin-mediated protein degradation

  • Protein complex that degrades cellular proteins by proteolysis
  • located in nucleus and cytoplasm

Functions

  • regulate protein levels and degrade misfolded proteins

Structure

  • 26S proteasome Mr 2000 kDa
    • two 19S regulatory caps -ATPase active sites and ubiquitin binding sites
    • one 20S core hollow structure – catalytic core

Ubiquitin

Calpain

Function

  • proteolysis in embryogenesis, regulation of key enzymes, structural proteins and in proinflammatory responses
  • mediates MMP2 expression and cell migration (fibroblasts and leukemic cells)
  • contribute to cell death in neurons by cleaving essential cytoskeletal proteins
  • tissue damage in response to pathological events (myocardial infarcts, stroke, atherosclerosis, and brain trauma)
  • Deregulated calpain activity following loss of Ca2+ homeostasis
  • intracellular free calcium concentration (Ca2+i)regulator in some late apoptotic signaling

Cell Stress

The term “cell stress” can cover many different issues. It is used here to describe two specific circumstances: when normal cellular processes may function abnormally (protein misfolding), or under specific limiting growth conditions (starvation). Under these conditions the cellular response is graded, initially to correct the problem (Unfolded Protein Response) or reuse existing resources (Autophagy).

Endoplasmic Reticulum Stress

  • Endoplasmic Reticulum functions
    • protein synthesis
    • lipid metabolism
    • calcium regulation (Ca2+) storage, release, signaling

Abnormal protein folding can lead to the Unfolded Protein Response (UPR)

  • accumulation of misfolded proteins
  • aggregate in the ER lumen
  • causes ER stress

Unfolded Protein Response (UPR)

  • decrease in the arrival of new proteins into the ER
    • preventing additional protein misfolding and overloading of the organelle
  • increase in the amount of ER chaperones
    • increasing the folding capacity of the ER to deal with misfolded proteins
  • increase in the extrusion of irreversibly misfolded proteins from the ER
    • subsequently degradation of these proteins in the proteasome

If all the above UPR steps fail, cell death by apoptosis is triggered.

Classification of Cell Death

Cornification

  • Elimination of cytosolic organelles
  • Modifications of plasma membrane
  • Accumulation of lipids in keratohyalin granules in stratum granulosum
  • Extrusion of lipids in the extracellular space
  • Desquamation (loss of corneocytes) by protease activation

Cornified envelope – formation or ‘keratinization’ is specific of the skin to create a barrier function. Although apoptosis can be induced by injury in the basal epidermal layer (e.g., UV irradiation), cornification is exclusive of the upper layers (granular layer and stratum corneum).

Autophagy

  • Lack of chromatin condensation
  • Massive vacuolization of the cytoplasm
  • Accumulation of (double-membraned) autophagic vacuoles
  • Little or no uptake by phagocytic cells, in vivo

Autophagic cell death – defines cell death occurring with autophagy, though it may misleadingly suggest a form of death occurring by autophagy as this process often promotes cell survival.

Necrosis

  • Cytoplasmic swelling (oncosis)
  • Rupture of plasma membrane
  • Swelling of cytoplasmic organelles
  • Moderate chromatin condensation

Necrosis – identifies, in a negative fashion, cell death lacking the features of apoptosis or autophagy. Note that necrosis can occur in a regulated fashion, involving a precise sequence of signals.

Apoptosis

  • Rounding-up of the cell
  • Retraction of pseudopodes
  • Reduction of cellular and nuclear volume (pyknosis)
  • Nuclear fragmentation (karyorrhexis)
  • Minor modification of cytoplasmic organelles
  • Plasma membrane blebbing
  • Engulfment by resident phagocytes, in vivo

Apoptosis – is the original term introduced by Kerr et al. to define a type of cell death with specific morphological features. Apoptosis is NOT a synonym of programmed cell death or caspase activation.

Cornification

Occurs in the skin epithelium and occurs in the upper layers (granular layer and stratum corneum)

  • Elimination of cytosolic organelles
  • Modifications of plasma membrane
  • Accumulation of lipids in keratohyalin granules in stratum granulosum
    • F-granules (histidine-rich) are large, irregularly shaped granules
    • L-granules (sulphur-rich)
  • Extrusion of lipids in the extracellular space
  • Desquamation (loss of corneocytes) by protease activation
  • Apoptosis can also be induced by injury to the basal epidermal layer
    • UVB irradiation, chemicals, cytotoxic cytokines

Autophagy

The Cell – Lysosomes in phagocytosis and autophagy

See also Lecture – Endocytosis

Autophagy Processes

There are several different classifications of autophagy this is probably the simplest.

  1. macroautophagy (also called autophagy)
  2. microautophagy
  3. chaperone-mediated autophagy
  • to remove abnormal cytoplasmic organelles and components, it is also a stress response
    • cellular self-catabolic process “eating oneself”
  • initial sequestered in a structure called a phagophore
  • which then closes into a double membrane vesicle the autophagosome
    • some autophagosomes formed in a PI3P-enriched (phosphatidylinositol 3-phosphate) membrane compartment dynamically connected to the endoplasmic reticulum
  • an autophagosome fuses with a lysosome
  • Regulated process of the degradation and recycling of organelles and cellular components
  • Resulting in organelle turnover and in the bioenergetics of starvation
  • Could result in cell death
    • through excessive self-digestion and degradation of essential cellular constituents

Some Recent Findings

  1. Autophagosomes are derived from mitochondrial outer membrane during starvation
  2. Lipids, but not most proteins, are transferred from mitochondria to autophagosomes
  3. Mitochondria-ER connections are required to form autophagosomes during starvation
  4. Mitochondrial contribution to autophagosome assembly is unique to starvation

Necrosis

  • Greek, nekros = corpse
  • pathological cell death from extrinsic injury
    • tissue damage
  • autoimmune insulin-dependent diabetes?
  • irreversible

Induction

  • tumor necrosis factor, double-stranded RNA, viral infection or bacterial toxins
  • does not shut down of protein synthesis
    • occurs in apoptosis due to caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-

Early stages

  • cell and organelles (mitochondria) swell (oncosis)
    • (Greek, onkos = ‘swelling’) previously described as a separate form of cell death
  • due to disruption of plasma membrane
  • cell contents leak out leading to inflammation and necrosis

Late stage

  • loss of cell membrane integrity
  • finally cell disintegration
  • cell lysis can also trigger an inflammatory response
    • leading to further inflammation and damage
    • triggering a cycle of death

Programmed Cell Death

Until very recently this has been exclusively about apoptosis.

There is a theory though that there is no such thing as “unregulated” cell death and that even necrosis may involve a regulated program. In addition, a novel cell death pathway has been identified in neutrophils in the fight against pathogens.

Neutrophil Extracellular Traps

Neutrophils in circulation are targeted by cytokines to migrate into infected tissues, where they activate, and engulf pathogens into a phagosome.

A second defense mechanism has recently been described Neutrophil Extracellular Traps (NETs)

  • composed of chromatin decorated with cytoplasmic proteins in the extracellular space
    • bind Gram-positive and -negative bacteria, as well as fungi

Pathway

  • requires generation of reactive oxygen species (ROS) by NADPH oxidase
  • lobulated nuclear morphology lost
  • euchromatin and heterochromatin distinction lost
  • all the internal membranes disappear
    • allowing NET components to mix
  • NETs emerge from the cell as the cytoplasmic membrane is ruptured
    • process distinct from necrosis or apoptosis

Apoptosis Signals

A diverse group of signals can induce apoptosis

Selective process for deletion of cells

  • Superfluous
  • Infected
  • genetic errors
  • transformed cells

Process required for

  • Embryogenesis
  • Metamorphosis
  • Endocrine dependent tissue atrophy
  • Normal tissue turnover
  • Variety of pathologic conditions

Apoptosis Examples

Development

Limb development

Ossification

  • Death of chondrocytes

Nervous System

  • Death of neurons
  • Disruption of Brain Development

Apoptotic Cell Morphology

The following cellular changes occur in sequence during apoptosis.

  • loss of cell membrane phospholipid asymmetry
  • Condensation of chromatin
  • Reduction in nuclear size JCB – Nucleus changes
  • Internucleosomal DNA cleavage TUNEL staining
    • DNA ladder
  • shrinkage of the cell
  • membrane blebbing
  • breakdown of the cell into membrane-bound apoptotic bodies (apoptosomes)
    • bodies then phagocytosed by other cells

A number of different experimental techniques have been developed to identify these changes. Apoptosis Methods

Two Main Pathways

  • death-receptor pathway (extrinsic)
  • mitochondrial pathway (intrinsic)
    • may also be several alternate pathways

Both pathways

  • Converge on caspase-3 activation
  • branch into many pathways
  • leading to eventual cell death

Death Receptor Pathway

Receptors

Cell surface receptors contain an intracellular death domain (DD)

  • belong to the tumor necrosis factor (TNF) super family
    • Fas (CD95/Apo1), TNF receptor 1 (p55), TRAMP (WSL-1/Apo3/DR3/LARD), TRAIL-R1 (DR4), TRAIL-R2 (DR5/Apo2/KILLER)
  • trigger apoptosis upon ligand binding

Ligand Binding

  • Fas Ligand (CD95 ligand) binds Fas
  • TNF and lymphotoxin a bind to TNFR1
  • TWEAK (Apo3 ligand) binds to TRAMP
  • TRAIL (Apo2 ligand) binds both TRAIL-R1 (Pan et al., 1997) and TRAIL-R2

Upon ligand binding receptor associates with an adaptor protein

  • Fas-associated death domain (FADD) directly or indirectly
    • through TNFR-associated death domain (TRADD)
  • FADD also interacts with pro-caspase-8
    • form a complex at the receptor called the death inducing signalling complex (DISC)

Death Inducing Signaling Complex (DISC)

  • DISC induces the activation of caspase-8
    • activates downstream effector caspases

Bid is also cleaved by pro-caspase 8 and translocates to the mitochondria to activate the intrinsic mitochondrial pathway, linking the two death pathways.

Decoy Receptors (DcR)

  • tumor necrosis factor (TNF) super family
  • inhibit death signaling by sequestration of ligand
    • DcR1, DcR2 and osteoprotegerin (OPG) bind to TRAIL
    • DcR3 binds Fas ligand

FLICE-like inhibitory protein (c-FLIP)

  • intracellular endogenous inhibitor
  • regulates by interacting with FADD
    • blocks apoptosis

Mitochondrial Pathway

  • begins with permeabilisation of mitochondrial outer membrane
    • either permeability transition (PT) pore dependent or independent

Permeability Transition (PT) Pore

Structure formed from matrix, inner membrane, outer membrane proteins

  • matrix protein cyclophilin D
  • inner mitochondrial membrane protein adenine nucleotide translocator (ANT)
  • outer mitochondrial membrane protein voltage dependent anion channel (VDAC)

Opening the PT pore

  • triggers the dissipation of the proton gradient created by the electron transport
    • uncoupling of oxidative phosphorylation
  • also causes water to enter the mitochondrial matrix
    • results in swelling of the intermembranal space
    • rupturing of the outer membrane
    • releasing apoptogenic proteins – cytochrome c, apoptosis inducing factor (AIF), endonuclease G

Apoptosome

  • Formed by Cytochrome c, apoptosis protease activating factor (APAF-1) and pro-caspase 9
  • complex promotes the activation of caspase 9
    • activates effector caspases that collectively orchestrate the execution of apoptosis
  • AIF and endonuclease G – DNA fragmentation and chromosomal condensation

Other proteins released upon mitochondrial outer membrane permeabilisation

  • Smac/DIABLO – second mitochondria-derived activator of caspases/direct IAP-associated binding protein with low pI)
  • Omi/HtrA2 (high temperature requirement A2), which antagonize IAPs thereby promoting caspase activation

Bcl-2 Family

PT pore independent mitochondrial membrane permeabilisation is regulated by Bcl-2 family members anchored to the mitochondria membrane by hydrophobic C-terminal

Anti-apoptotic members

  • Bcl-2 and Bcl-xL
  • Bcl-2 proteins protect integrity of mitochondria
    • prevent cytochrome C release
    • block caspase 9 activation

Pro-apoptotic members

  • two categories based on expression of Bcl-2 homology (BH) domains
  • Multi-domain proteins comprise BH domains 1-3 and include Bax, Bak, and Bok
    • form channels in outer mitochondrial membrane releasing intermembranal space apoptogenic proteins
  • BH3 only proteins consists of Bad, Bik, Bid, Puma, Bim, Bmf and Noxa.
    • activate multi-domain pro-apoptotic species
    • disrupt the function of anti-apoptotic Bcl-2 family members

Possible Bcl Mechanisms

  • Formation of a pore – cytochrome c and other intermembrane proteins escape
  • Heterodimerization – between pro- and anti-apoptotic family members
  • Direct regulation of caspases – by adaptor molecules
  • Interaction with mitochondrial proteins – generate a pore or to modulate mitochondrial homeostasis
  • Oligomerization- form a weakly selective ion channel

p53

Induced by pro-apoptotic stimuli activates apoptosis by intrinsic mitochondrial pathway

Normally p53 low levels

  • by murine double minute-2 (MDM2) or the human homologue (HDM2)
  • inhibit p53 transcription
  • promote p53 degradation (by proteosome)

Activation of p53

  • stabilization of p53 by post-translational modifications
    • disrupt interaction between p53 and MDM2
  • p53 drives expression of APAF-1 and pro-apoptotic Bcl-2 family members and transcriptional independent death pathways

Caspases

  • cysteine proteases
  • central regulators of apoptosis
  • 14 different caspases identified
  • expressed as pro-enzymes
    • three domains NH2 terminal, a large subunit 20 kDa and a small subunit 10 kDa
  • activation by proteolytic processing between domains
    • allows association of large and small subunits
  • active caspases are a tetramer 2 heterodimers of large and small subunits

Initiator caspases

  • caspase 8, 9, 10 and 12
  • closely linked to pro-apoptotic signals
  • once caspases activated cleave and activate effector

Effector caspases

  • caspases 3, 6, and 7
  • cleave cytoskeletal and nuclear proteins
  • induce apoptosis

FasL and Fas-expressing Cells

Caspase Cleavage

  • leads to diverse results
  • depending on substrate and position of cleavage site
  • loss of biological activity eg lamin network
  • limited proteolysis by caspases result in a gain of biological activity eg Bcl-2 or Bcl-x, PAK2, Bid and CAD/ICAD

Caspase Activation Mechanisms

(a) proteolytic cleavage by an upstream caspase activation of downstream effector caspases induction of non-caspase proteases (granzyme B)

(b) Induced proximity aggregation of procaspase-8 molecules somehow results in cross-activation

(c) holoenzyme formation cytochrome c and ATP dependent oligomerization of Apaf-1 recruitment of procaspase-9 into apoptosome complex Activation of caspase-9 mediated by conformational change

Apoptosis Inhibitors

  • directly inhibit caspases or prevent activation

Akt

  • serine/threonine protein kinase
  • important anti-apoptotic factor
  • inhibits pro-apoptotic Bcl-2 family member, Bad
  • directly inhibits caspase 9

TNF-alpha

  • Tumour Necrosis Factor -alpha
  • both anti-apoptotic and pro-apoptotic effect
  • activate the transcription factor NF-kB
  • which then induces expression of IAP
    • an inhibitor of caspases 3, 7,and 9

Neurotrophins

  • regulate apoptosis through protein kinase cascades
  • phosphoinositide 3-kinase/Akt
  • mitogen-activated protein kinase pathways

Final Stages of Apoptosis

  • The membrane enclosed cell fragments are phagocytosed by macrophages and other cells.

Other Cell Death Terms

Anoikis

  • (Greek, anoikis = “homelessness”)
  • probably death by apoptosis
  • A form of programmed cell death that occurs when cells loose contact with the extracellular matrix (ECM)
  • Integrin regulation of cell viability through their interaction with extracellular matrix
  • Protein kinase and apoptosis-related signals can control anoikis both positively and negatively
  • tumorogenesis protection

Oncosis

  • (Greek, onkos = ‘swelling’)
  • the process occurring in early necrosis

Pyroptosis

  • Greek, pyro = fire or fever; ptosis = falling
  • proinflammatory pathway
  • cell death mediated by the activation of Caspase-1
  • also activates inflammatory cytokines, IL-1ß, and IL-18

Chapter 16 Cell Communication pp 531-570


Signaling Mechanisms

  • Endocrine
    • Hormone (at a distance)
  • Paracrine
    • Locally
  • Neurotransmitter
    • Specific form of paracrine
  • Autocrine
  • Local (self)
  • Contact Dependent

Cell Communication

  • Contact Mediated
    • display molecules on cell surface
    • recognized by receptor on another cell
  • Non-Contact Mediated
    • chemical signal
    • nearby or at a distance

Common Signals Signals and Receptors Signal Transduction Model Signaling between Tissues Regulation of cells and tissues

Hormones secreted by one tissue to regulate function of other cells or tissues

Chemical Signal Types

  • water soluble
  • lipid soluble

Movie: Hormone Signaling

Extracellular Signal Steps

  • Signaling Molecule
  • Synthesis
  • Release by signaling cell
  • Transport to target cell
  • Detection by a specific receptor protein
  • Change by receptor-signal complex (trigger)

Cell Surface Receptors

  • four main classes
    • G protein–coupled receptors
    • ion-channel receptors
    • receptors linked to cytosolic tyrosine kinases
    • receptors with intrinsic catalytic activity
  • ligand binding to cell-surface receptor
    • trigger intracellular pathways
    • modulate cellular metabolism, function, or development
  • Removal of the signal
    • often terminates cellular response

Second Messengers

  • Cyclic nucleotides
    • cAMP, cGMP
  • Calcium Ions
  • Protein Kinase A
    • PKA, B, C
  • diacylglycerol (DAG)
    • modified lipid activates PKC
  • regulate the activity of cellular proteins
    • enzymes and non-enzymatic

Links: Cell – Common intracellular signaling proteins Elevation of cytosolic Ca2+ via the inositol-lipid signaling pathway | View Movie: Second Messengers in Signaling Pathways

Steroid Responses

Steroid_signaling_cartoon

Cytosol location

  • receptor bound to inhibitor
  • ligand binding activates receptor
  • translocates to nucleus on ligand binding

Nuclear location

  • binds ligand and DNA
  • becomes transcription factor

Steroid Hormone Receptors (SHRs)

Steroid hormone receptor family

  • estrogen receptor, two forms ERα [NR3A1] and ERβ [NR3A2]
  • cortisol binding glucocorticoid receptor (GR) [NR3C1]
  • aldosterone binding mineralocorticoid receptor (MR) [NR3C2]
  • progesterone receptor (PR) [NR3C3]
  • dihydrotestosterone (DHT) binding androgen receptor (AR) [NR3C4]

Steroid_signaling_cartoon

Nuclear Receptor Signaling

Steroid Receptors

  • steroid binding region
  • near C-terminus
  • DNA binding
  • central region
  • zinc finger motif
  • alpha helix and 2 beta sheets held in place by cysteine or histidine residues by a zinc atom
  • multiple fingers typical
  • DNA response element
  • Enhancer

Types

  • Type I Receptors
    • Sex hormone receptors (sex hormones) – Androgen receptor, Estrogen receptor, Progesterone receptor
    • Glucocorticoid receptor (glucocorticoids)
    • Mineralocorticoid receptor (mineralocorticoids)
  • Type II Receptors
    • Vitamin A receptor
    • Vitamin D receptor
    • Retinoid receptor
    • Thyroid hormone receptor
  • Orphan receptors

Eicosanoids

  • lipids acting as signaling molecules
  • act by binding to cell surface receptors
    • prostaglandins, prostacyclin, thromboxanes, and leukotrienes
  • rapidly broken down
  • act locally in autocrine or paracrine signaling pathways
  • synthesized from arachidonic acid

G Protein-Coupled Signal Pathways

Figure 20-6. Schematic overview of common signaling pathways downstream from G protein–coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs)

Activation of adenylyl cyclase following binding to a Gs protein – coupled receptor

G-protein-coupled receptor structureG-protein coupled receptors
  • Transmembrane proteins transduce extracellular signals
  • common structural motif of 7 membrane spanning regions
  • Receptor binding promotes interaction
    • between receptor
    • G protein on interior surface of membrane
  • induces an exchange of GDP for GTP on G protein α subunit and dissociation of the α subunit from the βγ heterodimer

GTP-α subunit complex mediates intracellular signaling, depending on isoform either

  • indirectly – by acting on effector molecules adenylyl cyclase (AC), phospholipaseC(PLC)
  • directly – by regulating ion channel or kinase function

Receptor associated with Kinase

  • many growth factors use this pathway
    • Vascular Endothelial Growth Factor
    • Epidermal Growth Factor
    • Nerve Growth Factor
    • Bone Morphogenic Protein
    • Transforming Growth Factor-beta

Pathway

  1. Ligand binding
  2. Receptor association
  3. Phosphorylation
  4. Kinase cascade

VEGF Receptor and Ligands (example) EGF Receptor Transduction Pathway Signaling Pathway of TGF-β

  • TGF-β receptor
  • include Type I and II subunits
  • are serine-threonine kinases
  • signal through SMAD family of proteins
  • binding of TGF-β to cell surface receptor Type II leads to phosphorylation of Type I receptor by Type II

TrkA Receptor

The PI 3-kinase pathway and cell survival

  • trk (often pronounced ‘track’) stands for tropomyosin-receptor-kinase (not tyrosine kinase or tropomyosin-related kinase)
  • proteins with a single transmembrane helix
  • Trk proto-oncogenes – TrkA, TrkB, TrkC, TrkE
    • variably expressed in CNS and PNS

TrkA Pathway

  1. following neurotrophin binding
  2. dimeric Trk receptors
  3. phosphorylate one another at tyrosine residues (Y490 and Y785 for TrkA)
  4. phosphotyrosines bind adaptor molecules such as Shc and PLC-γ.
  5. these signaling intermediaries activate three major signaling pathways
    1. Ras/MAPK cascade
    2. PI3K/AKT
    3. IP3-dependent Ca2+ release (?)
  6. pathway activation results in transcriptional changes of different target genes

Proto-oncogenes

  • Normal cell proteins that have potential to cause uncontrolled growth when mutated
    • loss of receptor regulation
    • cells grow out of control
    • mutation in TK Receptor – receptor always activated
    • mutation of activating protein – always active
    • Oncogenes – Ras
  • mutants detected in 30% cervical cancers

Movie: Methods Receptor/Ligand MCB ch20anim3.mov

Phosphatases

The mechanism for dephosphorylation is through phosphatases.

Three main families of phosphatases

  1. phospho-Tyr phosphatases (PTP)
  2. phospho-Ser/Thr phosphatases
  3. those that cleave both

Phosphatase specificity by binding protein cofactors

  • facilitate translocation and binding to specific phosphoproteins
  • active phosphatase consists of a complex
    • phosphatase catalytic subunit
    • regulatory subunit
  • Regulatory subunits for Tyr phosphatases may contain a SH2 domain allowing binding of the binary complex to autophosphorylated membrane receptor Tyr kinases.

Protein Tyr phosphatases (PTPs)

  • consist of receptor-like (transmembrane) and intracellular Tyr phosphatases (about 100 PTPs)

PTP1B – dephosphorylates many cell surface receptors (insulin, EGF, PDGF) that have been phosphorylated on Tyr residues

Microscopy Methods


This Lab is an introduction to cell biology methods using microscopy. It includes a brief historic background and relevant modern technological advances. The focus is more on the application of these techniques in cell biology, rather than a comprehensive understanding of the physics and technology underlying the techniques.

Microscopy Techniques

  • Light Microscopy
    • normal – transmitted brightfield illumination of fixed and stained specimens
    • inverted – overcome focal length problems, combine with special optical techniques
    • optics – Phase contrast, Nomarski Differential Interference Contrast (DIC)
  • Electron Microscopy
    • transmission
    • scanning
    • tunneling
  • Fluorescent Microscopy
  • Confocal Laser Scanning Microscopy (CLSM)
  • Total Internal Reflection Fluorescence Microscopy (TIRFM)
  • Live Cell Imaging Timelapse

Light Microscopy

Transmission Microscopy

Useful for fixed and histologically stained cells or tissue sections. Histology Stains

Phase Contrast Microscopy

  • refractive index differences within cellular components and between cells and their surrounding aqueous medium
  • enhances contrast in transparent specimens
  • “phase halo” – can be either bright around dark objects or dark surrounding bright objects
  • diffracted light passes through the phase ring as well as the nonphase areas and interacting at the image plane
  • light diffraction and interference and not of the optical path of the sample

Differential Interference Contrast (DIC) Microscopy

Used to observe structure and motion in unstained, transparent living cells and isolated organelles. This method produces a monochromatic shadow-cast image of optical phase gradient.

Polarized Light Microscopy

This generates structural anisotropy due to form birefringence, intrinsic birefringence, stress birefringence. For example, birefringent microtubules in the mitotic spindle.

Fluorescence Microscopy

  • Fluorescence The process where an atom or molecule is transiently excited by absorption of external radiation at the proper energy level (usually ultraviolet or visible light) to then release the absorbed energy as a photon having a wavelength longer than the absorbed energy.
  • Autofluorescence The generation of background fluorescence by endogenous metabolites and organic or inorganic fluorescent compounds present in cells (catecholamines, cytochromes, fatty acids, flavins, flavin proteins and nucleotides (FAD and FMN), lipofuchsin pigments, porphyrins, reduced pyridine nucleotides (NADH and NADPH), serotonin, vitamin B)

Confocal Laser Scanning Microscopy (CLSM)

See also Laboratory 7 – Confocal Microscopy

  • optical microscopy technique based on wide-field fluorescence microscopy
  • a laser beam is focussed into the sample and using electronic lenses and apertures (pinhole) only the fluorescence light that comes directly from the confocal plane is detected by a photomultiplier
  • fluorescence from outside this plane is cancelled out by a pinhole

Links: Introduction to Confocal Microscopy | Figure 9-19. Conventional and confocal fluorescence microscopy compared

Live Cell Imaging Timelapse

This technique views cells growing in culture by a video camera linked to an inverted phase microscope. The cells also need to be maintained at physiological temperature, usually by a heated stage or container and carbon dioxide level, either by a sealed tissue culture flask or gassed container. Note that light levels must be very low, or shuttered, and a still camera can also be set to take an image at regular intervals, these images can then be put together as a movie.

Preparation/Fixation


This lab is an introduction to histological techniques and tissue/cell fixation. The lab will also introduce Occupational Health and Safety (OHS) issues in relation to chemicals used in this process. More information is available from the School of Medical Sciences OHS webpage. Later analysis and immunhistochemistry will be covered in a future Laboratories.

It is critical to match the method of fixation with the intended analytical technique. Some types of analysis are totally incompatible with certain fixation techniques and always consider that “artefacts” can be introduced by the fixation process.

In general the Fixation process should:

  1. Preserve cell structure by prevention of tissue autodigestion (autolysis)
  2. Inhibits bacterial and fungal growth (preserves)
  3. Make the tissue resistant to damage during subsequent processing (hardy)

Three Main Techniques

  1. Fresh Frozen
  2. Precipitation
  3. Aldehyde Cross-linked

Fresh Frozen

  • cells are preserved and hardened by rapid freezing
  • Used in surgical biopsies of tissue
  • advantages and disadvantages
    • advantages – rapid processing, retention of some enzyme and protein function, retention of epitopes, retention of fat
    • disadvantages – requires a cryotome (freezing microtome) for sectioning, thicker sections (8+ micrometers), tissue distortion with cutting, thawing can degrades tissue

Precipitation

  • Immersion in cooled organic solvents- methanol or acetone or acids
  • Acidic precipitation does not preserve cellular structures well, rarely used (except for specific protocols, such as mitotic chromosome spreads)
  • Fixation by precipitation does not preserve the three-dimensional organization of specimens, therefore not recommended for confocal microscopy.
  • Cultured cells fixed with cold methanol shrink by as much as 50%.
  • Advantages- speed -(fixation usually taking a few minutes), retention of epitopes (antibody binding sites) not covalently modified as they might be with aldehyde fixation,

simultaneous permeabilization of cellular membranes (no need for detergent-treatment), precipitation will not introduce autofluorescence

(Text modified from Cell Biology Applications of Fluorescence Microscopy by Stephen Rogers)

Methanol

  • precipitation fixation
  • Methanol dehydrates, coagulates and precipitates cellular proteins, nucleic acids and carbohydrates
  • The process involves no covalent bonding between methanol fixative and tissue components

Chloroform-containing Fixative

  • Carnoy’s fixative
  • rapid tissue penetration (small tissue pieces in minutes not hours)
  • can damage tissues when transferred from aqueous solution (extreme hydrophobicity of chloroform and rapid dehydration)

Fixative components

  • Chloroform 30%
  • Ethanol (100%) 60%
  • Acetic Acid (Glacial) 10%

Aldehyde Cross-Linked

  • Aldehydes form covalent bonds between adjacent amine-containing groups through a Schiff acid-base reaction.
  • Cross-links are generated between several reactive groups (mainly -NH2 groups) such as found in protein lysine residues.
  • good fixatives for proteins and nucleic acids.
  • most commonly used aldehydes are formaldehyde (formalin), paraformaldehyde and glutaraldehyde
  • The degree of cross-linking produced in a tissue is also proportional to fixation time.
  • Aldehydes are suspected carcinogens, to be used only in well-ventilated areas or fume hoods and contact with skin or eyes avoided

Formalin

  • Aldehyde Cross-Link fixation
  • Formalin is a 37% aqueous solution of formaldehyde, which fixes by cross-linking like other aldehyde fixatives and is suitable for most histological purposes
  • Neutral buffered formalin (fixation time 12-24 hours) is preferred to formol-saline (a single 10% solution of formalin in 9% aqueous NaCl) as formalin pigment is avoided
  • Specimens may be stored in this fluid and the solution is isotonic.
  • Can be combined with a precipitation step (acetone etc) for permeabilization
  • Synonyms: bvf, FA, fannoform, formalith, formalin, formalin 40, formic aldehyde, formol, fyde, hoch, karsan, lysoform, methyl aldehyde, methylene glycol, methylene oxide, methanal, morbicid, oxomethane, oxymethylene, paraform, polyoxymethylene glycols, superlysoform
  • Molecular formula: CH2O CAS No: 50-00-0 MSDS: Formaldehyde MSDS

Paraformaldehyde

  • Aldehyde Cross-Link fixation
  • Used generally fresh
  • generates less fluorescent artifacts than formaldehyde

Uses: immunochemistry, in situ hybridization, cell staining

Synonyms: paraform, polyoxymethane, polymerised formaldehyde, alacide, flo-mor, formagene

Molecular formula: (CH2O)n CAS No: 30525-89-4

Gluteraldehyde

  • Aldehyde Cross-Link fixation

Other Fixation Considerations

Detergents

  • Detergents are not really “fixative”, but a number of different types are often used in the fixation process.
  • Detergents can selectively remove components from the material to be fixed or already fixed, as a method of preserving or accessing antigenic sites that may be blocked or effected by the fixation process itself.
  • The 2 major detergent classes
    • ionic detergents
    • nonionic detergents

Osmolality

  • Generally a phosphate buffered saline (PBS) is used but wil differ for some specific fixatives. Changes in osmolality can affect tissue structure and introduce artefacts.
    • hypertonic solutions may cause cells to shrink.
    • hypotonic solutions may cause the cells may swell and burst.

Tissue Embedding

Cell cultures

  • Cell cultures are usually only a layer or two of cells thick and are generally not embedded in a support media, except for electron microscopic (EM) preparation.
  • This tissue thickness also means that fixation can be quite rapid.

Paraffin Embedding

Automatic wax embedding machine

  • Paraffin waxes can allow easy long-term tissue storage and ease of sectioning by supporting the tissue during cutting.
  • Often requires a large number of steps in fixation, series of steps for embedding, sectioning and finally removal of embedding matrix for staining.
  • There are automated paraffin embedding systems that remove many of the preparation steps.
  • Can sometimes not be suitable for immunochemistry fluorescence techniques.

Cryoembedding

  • Possible freezing artifact, ice crystal formation if not controlled chilling. Freezing can be critical.
    • vapor phase of liquid nitrogen
    • thawing isopentane
  • OCT (Optimal Cutting Temperature) commercial Cryo Embedding Medium
  • Not suitable for large amounts, by volume) of tissue (usually 0.5 cm x 0.5 cm x 0.5 cm max)

Links: Face down cryoembedding technique

Immunochemistry


We have looked at microscopy techniques and how to grow and fix cells. Now we will begin to look at analytical techniques in cell biology. This laboratory is an introduction to immunological methods for analysis of cells and tissues in cell biology.

Before you Start

The simplest form of analysis involves looking at the cells following fixation, histological staining and analysis of microscopic images of cells.

This analysis includes quantification of: cell size, shape, specialized processes and number of cells.

  • cell size – can inform about cellular growth.
  • cell shape – can inform about cell differentiation, cell motility and cell death.
  • specialized processes – can inform about cell differentiation.
  • number of cells – can inform about proliferation, cell cycle and cell death.

Polyclonal Antibodies

Links: Biochemistry Figure 4.32. Polyclonal and Monoclonal Antibodies |

Monoclonal Antibodies

  • Myeloma is a bone marrow tumor that has been adapted to grow permanently in cell culture.
  • Fusion of myeloma cells with antibody-producing spleen cells.
  • Hybrid cell (hybridoma) can produce and secrete large amounts of a monoclonal antibody.
  • MCB Movie: Preparing Monoclonal Antibodies

Antibody Techniques

Immunofluorescence Labelling

Uses fluorescent labelled antibody or the anti-immunoglobulin antibody used to detect the intracellular location of proteins with the fluorescence microscope. (see also Microscopy Methods confocal microscopy, TIRF microscopy)

Fluorescence Activated Cell Sorting (FACS)

Uses fluorescent labelled antibody bound to the surface of living cells to identify and sort using a laser to detect the fluorescence.

Links: MBoC Figure 8-2. A fluorescence-activated cell sorter

Western Blotting

(immunoblot) – Uses a labelled antibody to specifically detect proteins separated by SDS polyacrylamide gel electrophoresis (SDS PAGE). Can also be modified as a dot-immunobinding technique, spotting a specimen directly onto a nitrocellulose membrane followed by reaction with monoclonal antibody and a biotin-avidin-peroxidase indicator system.

Links: Biochemistry – Figure 4.36. Western Blotting | MBoC Figure 8-18. Western blotting

Immunoelectron Microscopy

Uses antibodies to detect the intracellular location of proteins at high resolution by electron microscopy. Antibodies are labeled with gold particles and then applied to ultrathin sections, which are then examined in the transmission electron microscope (TEM). Gold particles of different diameters can be used to visualise two or more proteins simultaneously.

Links: Metallic Silver Deposit | EBS – Immunogold Labelling in Scanning Electron Microscopy | Immunogold labeling EM level

Enzyme-Linked ImmunoSorbent Assay

(ELISA, enzyme immunoassay or EIA )- Uses a labelled antibody to detect and quantify isolated proteins usually in a 96-well microtiter plate.

Links: Biochemistry – Figure 4.35 Indirect ELISA and Sandwich ELISA |

Antibody Microarray

Uses antibodies bound to a slide or substrate to specifically and quantitatively bind proteins from a cell extract (proteomic profiles), the protein levels in one sample are compared to those in a second sample.

Links: Clontech – Antibody Array

Immunochemistry Method

There are many descriptions of immunochemistry specific methods. The steps below give an example of some typical basic steps.

  1. Fixation – Cells/tissue section is fixed and if necessary permeablised.
  2. Blocking – with a protein solution to prevent non-specific binding of antibody.
  3. Primary Antibody Incubation – diluted to appropriate concentration in buffer or blocking solution. A range of times and temperatures can be used for this step e.g. 1 h at 37C, 2 h at RT, overnight at 4C.
  4. Washing – a series of washing steps to remove the excess primary antibody.
  5. Secondary Antibody Incubation – diluted to appropriate concentration in buffer or blocking solution. A range of times and temperatures can be used for this step e.g. 1 h at 37C, 2 h at RT, overnight at 4C.
  6. Washing – a series of washing steps to remove the excess secondary antibody.
  7. Mounting – there are a number of lab-made and commercial mountants that preserve either fluorescence or colour precipitate.

To download a PDF, click here.

Source: https://cellbiology.med.unsw.edu.au/cellbiology/index.php/ANAT3231_References

Formatted by: www.biolympiads.com