Cell Biology: Lecture 7 Notes

Actin Filaments

• Fine threads approximately $8nm$ in diameter.
• Composed of a tight helix of globular proteins known as g-actin.
• Polar, with a slow-growing minus end and a faster-growing positive end.
• Responsible for determining cell shape in many animal cells.
  • Give rise to projections such as villi, ridges, and furrows.
• Many cells that extend or contract contain actin arrays.
  • Examples: muscle fibers and pseudopodia.

Treadmilling vs. Dynamic Instability

• Actin and microtubules grow by regulating polymer length, followed by nucleotide hydrolysis (ATP or GTP).
• Actin Length:
  • Dependent on monomer concentration.
  • High concentration: grows at both ends.
• Microtubule Length:
  • Dynamic instability due to random GTP hydrolysis.
  • Grows/shrinks only at one end.

Actin Inhibitors & Other Facts

• CYTOCHALASINS: inhibit actin polymerization.
• PHALLOIDINS: bind to filaments and prevent depolymerization.
• Arrays of actin are bound together by linking proteins.
• The total length of actin in cells is about 30 times greater than microtubules.

Intermediate Filaments (IFs)

• A network of tough, durable fibers in the cytoplasm of most animal cells (approximately $12 nm$ in diameter).
• Surround the nucleus and form a coarse network that extends through the cytoplasm to the plasma membrane.
• Provide mechanical stability to animal cells; responsible for the shape of nuclei.
• Anchored to the cell membrane at desmosomes (cell-cell junctions).

Structure & Composition of IFs

• Not globular proteins but elongated fibrous structures.
  • Amino-terminal end (head).
  • Carboxy-terminal end (tail).
  • Intervening α-helical region.
• Assembled as coiled dimers, which aggregate in an antiparallel fashion into fibrous aggregates.

Intermediate Filament Tetramers

• Eight tetramers twisted together.
• Overlapping lateral interactions give strength.
• NO variation in the rod domain.
• Variation in the head domain amino acid composition allows interaction with different cytoplasmic components.

Strength from Intermediate Filaments

• Particularly prevalent in the cytoplasm of cells subject to stress.
  • Muscle cells.
  • Epithelial cells (skin).
  • Nerve cells – long processes.
• Fibers provide strength, like steel bars in concrete.

Four Classes of Intermediate Filaments

• CYTOPLASMIC
  • keratins: in epithelia
  • vimentin and vimentin-related: in connective tissue, muscle cells, and glial cells
  • neurofilaments: in nerve cells
• NUCLEAR
  • nuclear lamins: in all animal cells

Types of Intermediate Filaments

• Keratin ($40-70kD$):
  • In epithelial cells, forming hair and nails extracellularly.
• Vimentin ($54kD$):
  • In cells such as fibroblasts and white blood cells; often transiently expressed.
• Neurofilaments ($60-130kD$):
  • In the long axis of neurons, giving them shape.
• Lamins:
  • Underlie the inner face of the nuclear envelope.

Keratins

• Allow skin and other epithelia to stretch.
  • Example: gut epithelium during motility.
• Provide protection for the skin.
• Specialized keratin filaments:
  • Hair.
  • Nails.
  • Claws/feathers.
• Connected from one cell to another through desmosomes.
• Epidermolysis bullosa:
  • Caused by keratin gene mutations.
  • Results in vulnerability to mechanical injury.
  • Blistering, unable to withstand stress or mechanical force.

Nuclear Lamina

• A 2-D meshwork on the inner nuclear membrane.
• The intermediate filament protein here is lamin, which is less stable than keratin.
• Needs to disassemble and reform at cell division.
• Interacts with integral membrane proteins.
• Provides attachment sites for chromosomes.

Extracellular Matrix (ECM)

• Can surround cells as:
  • Fibrils that contact cells on all sides.
  • A sheet known as the basement membrane on which cells 'sit'.
• Provides:
  • Mechanical support.
  • A biochemical barrier.
  • A medium for extracellular communication, assisted by CAMs.
  • Stable positioning of cells in tissues through cell-matrix adhesion.
  • Repositioning of cells by cell migration during cell development and wound repair.

ECM Functions

• Provides tensile strength for tendons.
• Provides compressive strength for cartilage.
• Hydraulic protection for many types of cells.
• Elasticity to the walls of blood vessels.
• Each cell type has surface proteins that extend into the ECM or to the surface of other cells.
  • Determines cell properties and internal functions.
• Many cell surface proteins are linked to complex CHO modifications.
• Protein and sugar components are involved in adhesions between cells.
• The immediate area outside the cell is the glycocalyx.

ECM Origin

• Cells make ECM.
• ECM is specific to cell type.
  • Examples:
    • Fibroblast cells secrete connective tissue ECM.
    • Osteoblast cells secrete bone-forming ECM.
    • Chondroblast cells secrete cartilage-forming cells.
• Other forms:
  • Connective tissue has lots of ECM and not many cells.
  • Basal lamina: a tough layer containing many collagen fibers and laminin. Epithelial cells 'sit' on it.
    • Very little ECM around each cell.

ECM Roles

• Not just an inert scaffold; it provides mechanical support.
• With the glycocalyx, it provides:
  • A biochemical barrier.
  • A docking facility for imports and exports.
  • A medium for chemical signaling.
• Actively regulates cell behavior, influencing:
  • Shape.
  • Survival.
  • Development.
  • Migration.
  • Proliferation.
  • Function.

ECM Classes

Two main classes:

1. Polysaccharide chains – Glucosaminoglycans (GAGs) covalently linked to protein, proteoglycans forming gels
   • The repeating disaccharide sequence of a GAG Sulphate, carboxyl groups, negative charges

GAGs

• Form hydrated gels and occupy large amounts of space.
• Unbranched chains composed of repeating disaccharide units.
• Highly negatively charged due to sulphate or carboxyl groups on their sugars.
• Strongly hydrophilic, forming gels even at low concentrations.
• Water drawn in generates turgor.
• Allows compressive force to be applied.
  • Example: Cartilage matrix that lines the knee joint.

Proteoglycans

• Can Regulate The Activities Of Secreted Proteins
• Distinguished according to their linkage and number and location of sulphate groups
  1. Hyaluronan – no sulphate
  2. Chondroitin and dermatan sulphate
  3. Heparan sulphate
  4. Keratans
• Regulate movement of molecules and cells according to size, charge or both
• Bind secreted signal molecules, like growth factors, and enhance or inhibit signalling activity
• Binds and regulates activities of other secreted proteins Proteoglycans i.e. linked to protein

Hyaluronan

• Hyaluronan forms the backbone of complex proteoglycans such as aggrecan
  • Electron micrograph of an aggrecan complex from cartilage
• Hyaluronan molecules can have a length of up to $25,000$ repeats with a total mass of $10^6 Da$
• Hyaluronan is very flexible and twists and bend into many conformations
• Hyaluronan gives cartilage its gel-like properties

Fibrous Proteins

• which have structural and adhesive functions
• Collagen, elastin, fibronectin, laminin

Collagen Molecules

• Glycine, proline, hydroxyproline Triple helix (three fibres interwined together)
• The most abundant protein in humans making up from 25% to 35% of the whole-body protein content
• Type IV collagen is major structural component of the basal lamina
• Collagen contains the repeating tripeptide sequence: Gly-X-Y (X,Y: any amino acid, but are often proline and hydroxyproline
• Each polypeptide is twisted into a left-handed helix
• The three helices wrap around each other and produce a triple helix

Collagen IV Network

• Structure and assembly of the type IV collagen network
• Collagen IV contains globular domains at the N- and C-termini
• Globular domains can form multimeric interactions which results in assembly of a network
• Small non-helical regions introduce flexibility into the network
  • EM image of a collagen IV network

Fibrillar Collagens

• The extracellular matrix in connective tissues contains fibrillar collagens
• Type I, II and III collagen forms fibres ($80-90$ of all collagen in the body)
• Fibrillar collagen is the major component of tendon-rich tissue
• Association into fibrils is caused by hydroxylation of some proline and lysine side chains

Vitamin C Deficiency

• Vitamin C deficiency causes the disease scurvy
• Symptoms are spongy gums, loss of teeth, bleeding from mucous membranes, and finally death from bleeding
• Vitamin C is a cofactor of prolyl hydroxylase
• Incomplete hydroxylation of collagen prevents procollagen to assemble into normal fibers

Bone Composition

• Collagen is the major component of bones
• Bone is mostly made up from composite material of collagen and hydroxyapatite
• The bone mineral hydroxyapatite is a crystalline chemical arrangement of calcium phosphate $(Ca<em>{10}(PO</em>4)<em>6(OH)</em>2)$

Brittle Bone Disease

• Brittle bone disease (Osteogenesis imperfecta)
• Only $20-50$ of the normal amount of collagen being produced due to malformation
• Severe bone deformities
• Often results in stillbirth or death in the early years of childhood
• Collagen in the main organic component in the mineralized matrix of bones
  Mutation of glycine to any bulky residue in the Gly-X-Y sequence destabilizes the collagen triple helix

Elastin

• Stretching of a network of elastin molecules
• Elastin is highly hydrophobic
• Mainly two features: hydrophobic and cross- linked segments
• Hydrophobic segments provide elasticity
• Cross-linking provides stability

Fibronectin

• A glycoprotein dimer connected by disulphide bonds at the C-terminal end
• Can exist in soluble or fibrillar forms
  • Fibronectin (crucial for angiogenesis)

Fibronectins

• Fibronectins interconnect cells and the matrix
• Fibronectins help attach cells to the extracellular matrix by binding to other ECM components such as collagens and heparan sulphate proteoglycans
• Through interaction with adhesion receptors (integrins), fibronectin influences the shape and movement of cells

RGD Sequence

• The minimal sequence in the cell binding region which recognizes integrins is Arg-Gly-Asp (RGD sequence)
• The synergy region near the RGD sequence enhances integrin binding
  • Fibronectins interconnect cells and the matrix

Integrin Adhesion Receptors

• Integrin adhesion receptors mediate outside-in signalling

Integrin Activation

• Ligand binding induces conformational changes near the propeller and βA domain
• Integrin undergoes transition from inactive, bent conformation to active, extended conformation
• Activation involves separation of transmembrane domains and cytoplasmic tails
• The active form bind to adaptor proteins talin and kindlin

Integrins

• Integrins function as bidirectional signalling molecules
• Cell adhesion and migration and ECM assembly
• Ligand
• Integrin
• Outside-in
• signalling
• Inside-out
• signalling
• Cell polarity, survivial and proliferation,
• cytoskeletal structure and gene expression