Topic 3.3 - Cytoskeleton, cell cycle, cell junctions and ECM

Role of microtubules in cell cycle (2)

microtubules emanate from the centrosome and attach to the kinetochore of chromosomes

mitotic splindle aligns and segregates duplicated chromosomes spindle

role of actin in cell cycle (2)

actin filaments reorganise the cell to assume a spherical shape

after chromosomes are segregated, actin filaments form a actin-myosin contractile ring to pinch the cell into two

composition of actin filaments

actin monomers

meaning of actin monomers are polarised

each monomer has either ATP or ADP bound in deep cleft int he centre of the molecule

meaning of actin filaments are polarised

two ends of the actin filament polymerise at different rates

fast growing end = plus end

slow growing end = negative end

structure of helical polymer of actin (2)

all actin subunits have the same orientation such that the filament is polarised with a plus and minus end

helical polymer is stabilised by multiple contacts between adjacent subunits

in vitro assembly of actin filaments (3)

1. lag phase: assembly of nucleus is relatively slow → rate-limiting step

2. growth phase: monomers add to the exposed ends of the growing filament causing elongation

3. equilibrium phase/ steady state: growth of polymer balances shrinkage back to monomers

what facilitates differences in growth rates - actin filaments

changes in conformation of each subunit → ATP and ADP

actin molecules tightly bound to ATP => hydrolysed to tightly bound ADP soon after assembly into the polymer which reduces binding affinity for neighbouring subunits

forms of actin (2)

T form = actin carrying ATP → adds to filament

D form = actin carrying ADP → leaves the filament

purpose of actin filament cap

ATP cap stabilises polymer and prevents spontaneous disassembly

rate of addition can be faster than rate of bound ATP hydrolysis

explain actin filament treadmilling

polymer maintains constant length despite net flux in subunits in a static cell

actin binding proteins - profilin role

concentrates actin monomers at sites of assembly to promote filament elongation or maintenance

actin binding proteins - thymosin role

binds subunits and prevents assembly

actin binding proteins - Arp2/3 complex role (2)

nucleates assembly to form a branched network and remains associated with the minus end

allows subunits to bypass rate limiting step of filament nucleation

how are actin monomers bound to the cell membrane - nucleation promoting factors

Nucleation-promoting factors at the membrane have binding sites for profilin

Activation of nucleation-promoting factors leads to nucleation of branched actin filaments by ARP2/3 complex with aid of profilin

modes of cell migration - structures formed via actin (3)

stress fibres → contractile and exert tension

cell cortex → actin networks that enable membrane protrusion at lamellipodium

filopodium → spike like protrusions of plasma membrae that can sense extra-cellullar signals and allow a cell to explore its environment

mesenchymal cell migration - step summary

focal adhesions form behind protruding lamellipodia that connect to contractile stress fibres → brace the cell against the surface across which it is migrating and creates tension

mesenchymal cell migration - focal adhesions (2)

actin linked cell matrix junctions

link cell cytoskeleton in small transient patches to the extracellular matrix

functions of actin contractile structures (2)

cell membrane and muscle contraction

structure of skeletal muscle cells

form muscle fibres → huge multinucleated cells which form by fusion of many muscle cell precursors (myoblasts)

thick and thin filaments in a muscle fibre diagram

thick = myosin

thin = actin

structure of tubulin

alpha and beta monomers → each subunit comprises a tightly linked heterodimer

one tubulin subunit = alpha-beta heterodimer

structure of protofilament (2)

many adjacent tubulin subunits all with the same orientation

has a plus and minus end

structure of microtubules (2)

stiff, hollow tube formed from ring of 13 protofilaments aligned in parallel

polarised with plus and minus end

microtubules - dynamic instability meaning

microtubules depolymerise 100 times faster from end containing GDP tubulin than end containing GTP tubulin

microtubules - cap function

GTP cap favours growth → loss results in depolymerisation and shrinkage of microtubule

microtubule vs actin nucleation (3)

microtubule nucleation takes longer and is more complex

multiple tubulin heterodimers need to come together

additional protein is required to aid with nucleation

microtubule binding proteins - gamma tubulin role

nucleates assembly and remains associated with minus end of microtubule

microtubule binding proteins - katanin role

severs microtubules

microtubule binding proteins - MAPs role and types (2)

stabilise microtubules by binding along sides

tau = long branching structures

MAP2 = confined to cell body and dendrites

microtubule motor proteins - kinesin v dyneins difference

kinesins usually bind directly between cargo and the microtubule

dyneins require adaptor protein-like dynactin

microtubule motor proteins - kinesin v dyneins similarities (3)

both involved in intracellular transport and “walk” along microtubule track

both use ATP to fuel movement

usually “walk” towardsi plus end of microtubule

intermediate filaments - structure

monomer joins another monomer to form a dimer → central rod domains are aligned and wound together to form coils

two dimers can line up side by side to form antiparallel tetramer of 4 polypeptide chains → offset with respect to one another

tetramers pack together in rope-like arrow

intermediate filaments - role

provide mechanical strength to cells

importance of cel polarity during embryo development (2)

cells need to be orientated correctly when blastomere cells form epithelial sheet surrounding cavity

cell movement during gastrulation allows some cells to tuck into interior to form mesoderm and the endoderm while ectoderm cells stay on outside

importance of cell polarity during brain development

neurons in cerebral cortex develope from a common progenitor → proliferates on the inner surface of the cortical neuroepithelium to produce successive generations of neurons that migrate outwards

cytoskeleton - rac activation summary

activation of small GTPase rac leads to alterations in actin accessory proteins that promote formation of protrusive actin networks in lamellipodia and pseudopodia

cytoskeleton - rac activation effects (2)

promotes arp2/3 → branched actin network in cellular protrusion

decreases myosin activity → less contraction and formation of stress fibres

cytoskeleton - rho activation effects

increased myosin activity and nucleation of actin filaments → promotes formation of contractile actin bundles at rear of cell and assembly of stress fibres

how is polarity of a cell determined

by specific intracellular localisations of activated molecules

polarity of cells - rac and rho activation (2)

1. If cell detects chemoattractant, Rac will dominant at front of cell -> lead to growth of protrusive actin network

   • Second messengers within this pathway are short-lived -> protrusion is limited to region of the cell closest to the stimulant

2. Second signalling pathway is activated but in the rear of the cell -> triggers activation of Rho

Pathways are mutually antagonistic -> Rac promotes protrusion whilst Rho promotes actin-myosin contraction  -> directed movement of neutrophil

chemoattractant -def

a signal that the cell wants to move towards in a particular region of the cell

cell polarity - apical v basal domain of epithelial cells

apical domain is functionally distinct from basolateral domain

cell polarity - how are apical and basal domains of epithelial cells regulated

regulated by differential localisation of PAR and Crumbs in apical and Scribble in basolateral

Scribble and Par = mutually antagonistic

PAR and Crumbs reinforce each other

purpose of epidermis

provides waterproof barrier that is self-repairing and continually renewed

explain the epidermis is “stratified”

layers are different and follow an oderly progresion from the basal cell layer to the squamous cell layers

layers of skin (3)

epidermis → dermis → hypodermis

what cells in the epidermis differentiate

usually only basal cells

basal population - summary

small numbers of stem cells

large numbers of transit amplifying cells derived from stem cells

how are prickle sells anchored together

desmosomes via thick tufts of keratin filaments

what epidermal layer is water-proof

granular cells are sealed together to form waterproof layer

separates inner metabolically active layers from the outermost layer of eppidermis

keratinised squames - def and strcuture

dead cells whose intracellular organelles have disappeared

reduced to flattened scales filled with densely filled keratin

why doesn’t the epidermis peel off the dermis

overlying epithelium interdigitates with underlying dermis of connective tissue to provide further strength and support to epidermis

location of stem cells in columnar epithelia

crypts/ pits

apical v basolateral membrane of an intestinal epithelium cell

apical has active transporters to pump glucose from intestinal lumen in

basolateral has passive transporter proteins that facilitate diffusion of glucose into blood

types of cell-cell junctions (6)

tight junction

adherenes junction

desmosome

gap junction

hemidesmosome

actin-linked cell-matrix junction

tight junctions - fucntion

seals gaps between epithelial cells

usually at apical side

adherens junctions - function

connects actin filament bundles in one cell with that in the next cell (actin cytoskeleton)

actin filament bundles = tethered to cadherin by intracellular proteins → cadherins bind to cadherins on adjacent cells via homophilic interactions

desmosome - function

connects intermediate filaments in one cell to those int he next cell via non-classical cadherins

gap junction - function

allows passage of small water-soluble molecules from cell to cell

hemidesmosome - function

anchors intermediate filaments in a cell to extracellular matrix

actin-linked cell-matrix junction - function

anchors actin filaments in cell to extracellular matrix

adherens junctions - cadherins in presence vs absence of calcium ions

calcium ions bind to regions of hinges to prevent them from flexing or bendin → rigid, curved structure

absence of calcium allows for increased flexibility of hinge regions of cadherin repeats → floppier molecule is no longer orinetated correctly to interact with a caderin on another cell → adhesion fails

adherens junctions - how do intercellular cadherins interact

N terminal cadherins on cell 1 interact with C terminals of cadherin clusters of second cell

velcro-like connection binds cells together

adherens junctions - example of tissue remodelling

as pattern of gene expression changes, different groups of cells segregate from one another ccording to cadherins they express

ectoderm cells = E-cadherin

neural tube cells = N-cadherin

caderin-dependent cell sorting principles (2)

1. cells will segregate based on which cadherin they express

2. cells will segregate based on the level of cadherin they express

desmosomes - role in tissues (2)

provide mechanical strength to epithelial and distribute tensile forces in cells subjected to high mechanical stress

connected adjacent cells via associated keratin filament intermediate bundles

tight junctions - role in tissues (3)

prevent leakage of molecules across an epithelium

separate different membrane domains of an epithelium

contribute to polarity of epithelial cells

tight junctions - sealing strands

composed of transmembrane proteins that make contact across the intercellular space and create a tight seal

composition of each gap junction

cluster of homogenous intermembrane particles → each intramembrane particle corresponds to a connexon

gap junctions - connexon composition

composed of 6 connexin subunits → connexons can be homomeric or heteromeric

intracellular channels can be homotypic or heterotypic

2 connexons join across the intercellular gap to form a continuous aqueous channel connecting the two cells

what do gap junctions allow to pass (5)

ions

sugars

nucleotides

vitamins

cell signalling mediators

what dont gap junctions allow to pass (5)

porteins

nucleic acids

polysaccharides

anything larger than ~1.5nm in diameter

proteoglycans - composition

proteins and GAGs (large long, unbranched, highly charged sugars)

fibrous proteins - types (2)

collagen

elastin

glycoproteins - structure

proteins with short, branched, N-linked oligosaccharides

formation of glycoproteins

small sugars, folding and quality control in ER → Golgi

majority of processing in ER

formation of proteoglycans

massive GAGs

ER → Golgi

majority of processing in Golgi

composiution of matrix

tough, fibrous proteins embedded in polysaccharide gel-like material

matrix function

facilitates resistance to tensile and compressive forces

extracellular matrix can withstand being stretched

how are connective tissues different from each other

related to different composition and arrangement of matrix

how are connective tissues different from each other - examples (3)

Tendon = numerous fibrous proteins with little ground substance

Bone = calcified ground substance and fibrils

Cartilage = large amount of polysaccharide gel

what cellular activities does the extracellular matrix regulate (5)

Cell proliferation

Cell survival

Cell migration

Cell shape

Cell function

key macromolecules of extracellular matrix (3)

proteoglycans and GAGs

fibrous proteins

glycoproteins

origin of organic compounds of the amtrix

specialised fibroblasts = major ECM secreting cell in many connective tissues

eg. osteoblasts in bond and chondroblasts in cartilage

ECM composition - proteoglycans and GAGs

form gel-like ground substance which resists resistance due to high water content

proteoglycans take up 90% of extracellular space due to high water content but only 10% of dry weight of fibrous proteins

cause of proteoglycan and GAGs high water content

negative charge of GAGs attracts cations especially sodium → cations draw water into tissues via osmosis

what molecules do proteoglycans and GAGs allow diffusion of

nutrients, metabolites, hormones, growth factors

proteoglycan composition

most GAGs are attached to proteins as proteoglycans

proteoglycans can consist of multiple proteins and GAGs

fibrous proteins - collagen provides…

tensile strength → tissue can withstand stretching

fibrous proteins - structure of collagen (4)

1. single collagen peptide composed highly of proline and glycine = arranged into repeats as an alpha-helical polypeptide chain

2. three alpha chains come together to form collagen molecule → super helix/ triple-stranded collagen molecule

3. many collagen molecules assemble together via covalent cross-linking to form collagen fibrils

4. many collagen fibrils pack together to form collagen fibres

true or false: all collagen form fibres

false

collagen 4 forms sheet-like networks

collagen 9 decorates fibrillar collagens and mediates fibril interactions

how are collagen fibres formed (3)

1. Collagen precursors or procollagens = synthesised in cytosol of the cell and translocated into the ER lumen

   • Contains unstructured propeptides at either end -> prevent collagen fibrils from assembling within the fibroblast

2. Once procollagens have been secreted from the cell via exocytosis, extracellular procollagen proteinases remove terminal propeptides -> produce mature collagen molecules

3. Collagen molecules can aggregate into ordered collagen fibrils → fibres

fibrous proteins - elastin role

provides elasticity to the tissue or ability of tissues to be stretched

important for tissue like blood vessels ad lungs

fibrous proteins - elastin composition

composed of crosslinked network of elastin molecules and glycoprotein fibulin which provides support for the elastin molecules

glycoprotein - types (2)

fibronectin

integrins

glycoproteins - structure of fibronctin

contains numerous copies of 3 different fibronectin repeats → type 1, 2 or 3

2 type 3 repeats near c terminus contain important binding sites for cell surface integrins

other fibronectin repeats involved in binding fibrin, collagen, heparin

glycoproteins - role of integrins

facilitate binding via the cell skeleton and the ECM whre fibronectin is a ECM protein

glycoproteins - role of fibronectin

can sense and respond to intracellular tension conveyed through actin cytoskeleton and via integrins

type 3 repeats unfold when fibronectin is stretched → exposes cryptic binding sides that interact wit other fibronectin molecules to form fibronectin filaments

glycoproteins - laminin role

has multiple binding sites for proteins → important in organising basal lamina and other ECM components and anchoring them to cells via integrins