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Cytoskeleton - Coggle Diagram
Cytoskeleton
Cell movement
it starts with the extension of membrane, driven by actin assembly
Leading edge
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Branches are nucleated by side-filament binding Arp2/3 complex (which is activated by a nucleation promoting factor)
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NPFs
WASp is an NPF, it exists in a folded inactive form making the WCA domain unavailable, and its activation requires PI(4,5)P2 and binding to Cdc42 at RBD region of WASp (this coincidence detection prevents accidental activation)
WAVE also has a WCA domain that can activate Arp2/3, and is also activated by coincidence detection (binding to acidic phospholipids and Rac1)
Arp2/3
when inactive, Arp2 and Arp3 are in wrong configuration but when nucleated, Arp2 moves allowing complex to bind to side of preexisting filament
Actin subunits are brought in by WH2 domains of NPF and they bind to Arp2/3 template to nucleate filament assembly
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Adhesions
contractile actin bundles puts tension on focal adhesions, and integrins bind to actin network in contractile bundles. This moves the cell contents relative to the focal adhesion
Integrins are teh membrane proteins within a focal adhesion that connects actin bundles to ECM, integrins have an external fomain that binds to ECM (like fibronectin and collagen)
The cell attaches to the substrate, this means teh lamellipodium (the thin, sheet-like membrane protrusions found at the leading edge of motile cells) do not retract and allows forward movement
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formins
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this complex can nucleate actin assembly by binding 2 actin subunits so that +end is toward the FH2 domains, the filament can grow at + end
The FH2 dimer can bind to 2 terminal subunits then pivots between them allowing addition of new subunit then binding to it
FH1 domain is rich in proline residues which serve as binding for profilin by acting as a landing site for profilin-ATP-G-actin to increase local concentration of these complexes, the actin from these are fed into FH2 domain
Regulation
many exist in folded, inactive forms due to C terminal interactions with rest of protein
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Endocytosis
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Phagocytosis
Each antibody has a Fab domain that binds specifically to its antigen, as it coats the bacterium, the Fab domains interact with the cell surface antigen, this exposes teh Fc domain (in opsonization)
White blood cells have an Fc receptor that binds to Fc domain, this binding signals cell to engulf bacteria
Assembly of microfilaments at the site of interaction act with myosin motor proteins to force bacterium in
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recycling
The membrane required for lamellipodium extension is provided by internal endosomes as membrane recycles back to cell surface
adhesion molecules in focal adhesions at rear of cell are internalised and transported by an endocytic cycle to front of cell
Actin Organisation
The proteins Cdc42, Rac and Rho control are small GTPases, so when GDP-bound they are inactive, when GTP-bound they are active
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Activation of Cdc42 stimulates actin assembley by Arp2/3 using WASp resulting in formation of filopodia
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Activation og Rho activates a formin for unbranched actin filament assembly and phosphorylates myosin light chain using Rho kinase
Cdc42 has also been found to be crucial for cell polarity, in animals it binds to Par6; external signals are transmitted to Cdc42 which orientates cell with high Rac at front (to form leading edge) and Rho at rear (induces contractile structures)
Actin
Function
required for cell movement, cell division, vesicle transport, phagocytosis and organelle movement
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Organisation
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microvilli
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lateral attachments to plasma membrane are provided by ezrin-radixin-moesin (these are activated by phosphorylation by kinase which requires PI(4,5)P2 for activation)
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Structure
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actin polypeptide folds into 4 subdomains that generates 2 lobes separated by a cleft (which binds to ATP)
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F-actin
asssembled from G-actin monomers, each actin molecule contains a Mg2+ ion complexed with either ATP or ADP. G actin is 2 lobes separated by a deep cleft that has an ATPase fold at the bottom
the - end is the one with the exposed cleft; the + end is favoured addition of actin and - end favoured removal
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the initial formation (nucleation) is slow then rapid elongation, but as concentration of G monomer drops a steady state is reached
can make up 10% of total muscle cell protein weight, and in microvilli local actin concentration can be as high as 5mM
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Treadmilling
Critical concentration
the concentration at which the addition at one end is balanced by the loss at the same end, so it is the minimal concentration of monomers at which filaments form
above Cc there is net addition, below Cc there is a net loss
The + end has lower Cc than - end (0.6um to 0.12um) so has a faster binding constant so will grow at a lower monomer concentration
After ATP-actin is added to plus end, it is hydrolysed to form ADP-Pi-actin (and the Pi is more slowly released)
the Pi is slowly released so that toward the - end the actin contain ADP-G-actin, and + end is composed of ATP-actin or ADP-Pi-actin
This results in small conformational changes in actin which changes binding kinetics (ADP-actin binds less strongly to other actin monomers which explains why -end loses actin)
It is important to maintain cell shape, otherwise the cell membrane would be pushed forward
Phases
Nucleation phase- lag period in whihc G-actin subunits combine into an oligomer of 2-3 subunits, but then acts as a seed
Elongation phase- short oligomer rapidly increases in length by monomer addition at both ends (so as F-actin grows, G actin monomers decreases until equilibrium)
steady state phase- G actin monomers exchange with subunits at filament ends, but there is no net change in total filament length
rate of addition of ATP-G-actin is 10x faster at + end than - end (12/uM/s to 1.3/uM/s, so 1uM of ATP-G-actin added means 12 subunits added to + end and 1.3 to - end)
Binding proteins
Cofilin
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it binds by bridging 2 actin monomers and inducing a small change in twist of filament, the small twist destabilises the filament between with and without cofilin
by breaking the filament this way it generates many -ends which greatly increases net disassembly of actin
Profilin
binds to ADP-actin and changes the shape so released ADP and allows ATP to rebind by opening the cleft
it binds G-actin on side opposite nucleotide binding cleft,
ATP readily binds to G-actin (forms profilin-ATP-actin complex) which cannot bind to -end (since profilin clocks sites o G actin for -end assembly) but the complex binds to + end
Once new actin subunit subunit binds, profilin dissociates. Profilin activity does not enhance treadmilling but does ensure constant supply of ATP-actin formed from released ADP-actin
Thymosin β
stops addition of ATP-actin so controls concenctration of free monomer (so cell can hold a store in case it needs ATP for movement)
G-actin levels can be as high as 100-400uM but critical concentration is 0.2uM but no polymerisation because of Thymosin B4
example, platelets: 550uM of actin (220uM in unpolymerized form), and 500uM of thymosin-B4; when some free actin is incorporated into actin filaments then more actin-thymosin B4 will dissociate thus maintaining a steady supply of actin subunits available for polymerisation
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gelsolin and cofililin
Gelsolin is regulated by Ca2+ since when it binds it changes conformation allowing binding on side of actin and inserting between subunits causing breaking
spectrin
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Erythrocytes has a 14 subunit actin based network under the plasma membrane stabilised by tropomyosin and tropomodulin on -end, these short filaments allow binding of 4-6 spectrin molecules
spectrin attach to membrane via ankyrin (which binds to transmembrane bicarbonate transporter) or via band4.1 (this binds F-actin and glycophorin C
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Capping proteins
CapZ binds to +end of actin which inhibits addition or loss, adn its concentration is enough to cap any + end
regulating capZ
inhibited by phosphatidylinositol 4,5-bisphosphate
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Myosin
types
myosin I- single head, membrane associated and endocytosis; since they only have one head each, several must work together to generate movement
myosin II- they form bipolar filaments in which tail domains assemnle into thick bundles with cluster of head domainscontraction
myosin V- have 2 heavy chains so motor with 2 heads, long neck regoin with 6 light chains that bind to organelles and transport them
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Structure
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ATPase activity and F-actin binding site reside in S1; and S1 has actin-activated ATPase activity (since activity increases with actin presence), S1 fragment consists of the head and neck domains with associated light chains and S2 and LMM make up tail domain
essential light chain (stability), regulatory light chain (controls myosin activity via Ca2+ and phosphorylation), heavy chain tail (variable and determines binding), 6 proteins total
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myosin II can be cleaved by gentle treatment with chymotrypsin to yield heavy and light meromyosin, the heavy can be furthered cleaved by papain to yield subfragmnet 1 and subfragment 2
Power stroke
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Myosin head hydrolyses the ATP (ADP and Pi remain bound) and allows myosin head to move along the actin
hydrolysis of ATP provides energy to rotate the head with respect to th eneck domain, it moves into the cocked position and rebind to F-actin
the Pi released tightens actin binding which triggers the force-generating change of conformation to move the actin filaments relative to myosin
Ca2+ (released from sarcoplasmic reticulum) changes shape of tropomyosin and troponin to uncover the myosin binding sites
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This model is supported by the evidence that the distance myosin moves along the actin (myosin step size) is proportional to the length of the neck domain
myosni II takes 8nm discrete steps and generates 3-5piconewtons of force, it also only spends about 10% of each ATPase cycle in contact with F actin (duty ratio); myosin V takes many 72nm steps without releasing actin (so moves processicely) and has duty ration of 70%
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Microtubule
Structure
Tubulin
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Each subunit can bind to 1 GTP, in a-tubulin this is never hydrolysed, but is by B-tubulin and GDP is exchanged for GTP
MTOC
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centrosome is the main MTOC in animal cells, +end moves toward the cell periphery, -end is anchored in centrosome
during mitosis, cells reorganise microtubules to form bipolar spindles extending from spindle poles
In neurons, microtubules can form axons which transport organelles and have same polarity or dendrites which have mixed polarity
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centrosome structure
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surrounded by pericentriolar material, including gamma-tubulin ring complex
the complex nucleates microtubules and is attached at many positions around nuclear envelope; recent work shows protein complex augmin complex can bind to sides of existing microtubules then recruit the y-tubulin complex and nucleate assembly
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y-tubulin ring complex consisits of many y-tubulin, the complex acts as a helical template to bind aB-tubulin for new microtubule formation
a microtubule is composed of 13 laterally associated protofilaments (dimers) that form a tubule with 25nm external diameter
As the dimers are all orientated the same, each protofillament has an a-subunit at one end and B-subunit at the other; the B end is +end and a is -end
Function
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depending on function the MTOC can be centrosome, spindle poles or basal body
axonal transport
Experiments have shown that organelle transport can occur in both directions (Anterograde is cell body to axon, used in axon growth and delivery of vesicle, retrograde is moving recycled membrane to cell body)
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Kinesin-1
it is a dimer of 2 heavy chains each associated with light chain, it contains pair of N terminal head domains connected by linker domain to central stalk and in end in tail domains
head domain binds microtubule and ATP, linker domain used in forward movement, stalk used in dimerization through coiled-coil interaction of heavy chains , tail domain responsible for receptor binding
It is a +end directed microtubule motor protein and it mediates anterograde transport of organelles along axon
there is variation in family: kinesin 2 has 2 different heavy chain motor domains and third polypeptide that binds cargo; kinesin 5 has 4 heavy chains that crosslink and slide past one another; kinesin 14 is only family known to move to -end; kinesin 13 have 2 subunits with kinesin domain in middle of polypeptide
Activity
Kinesin1 head binds microtubule when ATP-bound, since the motor has a nucleotide free landing head which is strongly bound to protofilament and ADPbound trailing head which is weakly associated to protofilament
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the swinging motion causes linker domain to rotate forward which throws trailing head into position to become leading head
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Interaction with leading head and microtubule causes leading head to release ADP (creating necleotide free state that binds to microtubule) and it induces trailing head to hydrolyse ATP so now weakly bound to microtubule
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Dynein
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it is a 2-headed molecule built around 2 heavy chains, each heavy chain has a stem which binds other dynein subunits, linker domain is involved in motor activity
The head contains the AAA ATPase domain, which has a stalk between the fourth and fifth AAA repeats
When no nucleotide, linker binds to microtubule, when AT Pbinds it dissociates from microtubule and linker bends and now crosses second and third AAA repeats
interaction with microtubule, ATP hydrolysis and release of Pi causes linker to straighten which moves the cargo towards the -end. ADP is released and motor head remains bound, when ATP binds it releases and cycle repeats
dynactin
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Arp1-containing domain binds cargo, p150Glued is a long protein that contains dynein binding site and has microtubule binding site at one end
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Dynein is associated to + end via dynactin-EB1 interaction and held in inactive form, when microtubule reaches cell cortex it encounters activator
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Dynamics
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catastrophe
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The assembly end of the microtubule contains slightly curved nascent protofilaments, these protofilaments contain GTP-B-tubulin which zip up by lateral interactions (forming straight protofilaments), the growing end is then said to have GTP cap. A shronking microtubule has ends of GDP-B-tubulin, so if GTP in terminal B-tubulin become GDP then blunt end will curl causing catastrophe
The lateral protofilament-protofilament interaction in GTP cap are strong enough to prevent unpeeling at other end, the energy from hydrolysis is stored stored in the lattice as structural strain which explains why losing the cap causes catastrophe
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At + end, there is addition of GTP-B-tubulin (this generates GTP cap_, the elongated protofilaments have lateral interactions and straighten, this transition induces tension.
The interaction between B-subunit and a-subunit enhances GTP hydrolysis forming GDP-Pi-B-tubulin, the Pi is slowly released so microtubule goes GTP cap to GDP-Pi-B-tubulin then GDP-B-tubulin
Intermediate filament
Unique properties
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tensile strength, like hair and nails
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Subunit dimers
the dimer is held together through rod domains which associate as coiled-coil and 2 dimers associate and make tetramers which then interlock to form protofilaments
4 protofilaments associate into a protofibril, 4 protofibrils associate to generate filament
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Classes
keratin (class I and II)
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dimers form consisting of one basic keratin chain and one acidic chain, rod domains make a coiled coil
hard keratins are rich in cysteine residues that become oxidised to form disulphide bridges, and soft keratins are found in epithelial cells
Basal layer of cells proliferate and give rise to keratinocytes which have abundance of cytokeratins (soft keratin), the cytoskeratins associate with specialised attachment sites; keratinocytes eventually die creating a dead cell later
desmin (class III)
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In skeletal a lattice is formed of bands of desmin filaments that encircle the Z disc and are crosslinked to plasma membrane, th e lattice is attached to the sarcomere through interactions with myosin thick filaments. Desmin plays role in maintaining structural integrity
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lamins (class V)
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it is encoded by 3 genes: 1 alternately spliced forms lamins A and C, the 2 others encode B1 and B2
B lamins are expressed in all cells, where A and C are developmentally regulated; B lamins are post-translationally prenylated helping association with inner nuclear envelope membrane
They contain coiled coil regions (like all intermediate filaments) but also a nuclear locaisation sequence that targets them to the nucleus
They provide rigidity since the lamin meshwork is associated with chromatin on one side and attached to cytoskeleton on other; proteins like lamin-B receptor and emerin are on inner nuclear membrane and can bind to both chromatin-associated proteins and lamins
Attachment to cytoskeleton through inner and outer nuclear membranes invovles proteins with SUN and KASH domains: SUN domain proteins are synthesised on ER with SUN domain in ER lumen which are then transported through nuclear pores and associate with nuclear lamina; KASH domain proteins (like Nesprins) can associate with SUN domain in perinuclear space which then associate with intermediate filaments , actin and microtubules thus linking to cytoskeleton
They are broken down by phosphorylation by mitotic CDKs (due to the C-termianl prenylation, lamin B dimers remain associated with nuclear membrane)
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Actin-Myosin
Skeletal Muscle
Sarcomere
The actin are assembled with +end embedded in Z disc so that 2 sets of actin filaments in sarcomere have opposite orientations
During the crossbridge cycle, ATP hydrolysis is coupled with myosin movement to Z disc (the +end of actin), because myosin is bipolar its action draws the thin filaments towards the centre which shortens the centre of the sarcomere
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Scaffolding proteins
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nebulin extends along the actin all the way from Zdisc to tropomodulin, it consists of a repeating domains that bind to actin
titin has its heads associated with Z disc and extends to M band where another titin molecule extends to other Z disc; its role is to hold myosin in middle of sarcomere and prevent overstretching
Ca2+
cytosol concentration is below 0.1uM, usually maintained by Ca2+ ATPase which pumps Ca2+ from cytosol into sarcoplasmic reticulum
arrival of nerve impulse at NMJ triggers AP to travel down sarcolemma which then travel sdown invaginations called transerve tubules
this stimulates opening of voltage-gated Ca2+ channels in SR membrane which raises cytosol Ca2+ concentration
this concentration changes tropomyosin and troponin so they no longer block myosin binding (this regulation is thin-filament regulation)
tropomyosin is associated to troponin, troponin is a 3 subunit complex (TN-T, TN-! and TN-C), binding of Ca2+ to TN-C triggers movement of tropomyosin to expose binding sites
Nonmuscle
they contain several types of contractile bundles, these are similar to sarcomeres but less organised and lack troponin regulatory system
Contractile bundles
most commonly found as circumferential belt, they encircle inner surface of cell at adheren junction and are involved in maintaining epithelium integrity
can form stess fibres, these are along lower surfaces of cell or ECMs; their ends terminate at integrin containing focal adhesions
they can form contractile ring, which is a transient structure that assemble at dividing cell equator, as the ring contracts it pulls the plasma membrane in and divides cytoplasm
Smooth Muscle
contraction regulation
When myosin regulatory light chain (LC) is not phosphorylated, myosin II folded into inactive state
when it becomes phosphorylated by myosin lightchain kinase, myosin II opens up by unfolding and assembles into filaments
MLC kinase is regulated by Ca2+ in cytoplasm by binding to calmodulin, teh Ca2+/calmodulin complex then binds to MLC kinase and activates it (thick filament regulation)
stimulation
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norepinephrine, angiotensin, endothelin, histamine etc, can modulate or induce contraction of smooth muscle or elicit cahnge in shape and adhesion of nonmuscle cells