Lecture 3 - The cytoskeleton Flashcards
What are the functions of the cytoskeleton?
- cell morphology
- migration
- elongation
- chromosome separation
- vesicle transport
What are the three componants of the cytoskelteon?
- microfilaments (7-9nm)
- intermediate filaments (10nm)
- microtubules (24nm)
What are eight features of a migrating cell?
- actin bundle
- lamellepodia
- filopodia
- focal adhesions
- tail
- microtubule organising centre
- stress fibres
What are lamellopida?
broad membrane extensions that move forward and are typical of fastly migrating cell
What are filopodia?
fine cytoplasmic extensions
typical of slower moving cells
What are membrane ruffles?
assemblies of cytoplasm not tighly adheared to a substrate
What does an actin polymer consist of?
-consist of globular monomers of G-actin which nucleate and elongate in the same direction to form a filamentous helical polymer
What is the orientation and structure of the monomeric G-actin?
- Denatures in the absence of ADP or ATP
- Has an Mg2+ molecule bound
- Has orientation = ATP binding cleft orientatated towards top (-) end
How many subunits of G actyin make up one repeating helical turn?
28 G-actin subunits
72nm long
What is actin polymerisation dependent upon?
the equilibrium between concentration of monomers in cell and active elongation/severing
How does the critical actin concentration (Cc) affect actin formation?
Cc determines whether the filament can extend
- Under steady state conditions the dissociation rate = association rate (ATP hydrolysis to ADP is slower than the opposite reaction)
- During elongation the G monomer concetration is higher than critical concentration, and ATP is hydrolysed to ADP
- During dissasociation the G monomer concentration is lower than the Cc
What is involved in the active process of actin length regulation?
Severing and capping proteins
-interact with filamentous actin and cap and/or severe
Give an example of a severing/capping protein
Gelsolin - caps and severes (+) end CapZ - caps (+) end Tropomodulin - caps (-) end Cofilin - severes gCAP39 - caps (+) end Severin - caps and severes
How can capping and severing proteins alter the actin cytoskeleton?
- protect it by capping and consequently stabilising
- shortern by severing
- can cap at both ends so that it is permenantly stabilised
What are the features of Gelsolin?
- capping and severing protein that can help to regulate actin filament length alongside Cc
- activated by high Ca2+ concentration leads to a conformational change e.g. when growth faction binds to it’s ligand
- conformational change allows it to bind to the F-actin polymer and disrupt the subunit organisation causing severing
- then binds to the (+) end of the actin fragment
What is the structure of contractile bundle (stress fibres and cytoplasmic contractile ring) and what protein are they crosslinked by?
- contractile bundle with a loosely packed, antiparallel organisation (allow contraction by myosin II)
- crosslinked by alpha actinin
What is the structure of filopodia and what protein are they crosslinked by?
- tightly packed bundles of parallel F-actin (prevents myosin II binding)
- crosslinked by fimbrin
What is the structure of the gel-like network of actin in the cell cortex and what is it crosslinked by?
- gel-like network of randomly orientated fibres, provides structure
- fibres crosslinked by filamin at right angles
- required for lamellipodia formation and cell migration
What are four crosslinking proteins and what are their structures?
Fimbrin - monomer (filopodia)
alpha-actinin - dimer (stress fibres)
Filamin - dimer with a ‘hook like’ structure (network of fibres)
Spectrim - tetramer (red blood cells)
What does the branching of actin filaments require and what are these proteins?
Actin-related proteins (ARPs)
- complexes that can interact with actin and different actin binding proteins
- bind and act as a nucleation point for the formation of other fibres/filaments
Give an example of an ARP (actin relatied protein)
Arp2/3 complex
Binds at 70* to the side of an actin microfilament to nucleate daughter filaments
-dynamic and changes often in response to other things in the cell
What are mysosins, what are the three?
- multi gene family
- composed of 1 or 2 heavy chains and several light chains (may have a refulatory role in the action of the proteins)
- Myosin I = monomer
- Mysoin II and V = dimers
- bind to actin via head section which has ATPase activity and can generate force to walk along actin filament
What is the structure of Myosin I?
- monomer
- globular head region with ATPase activity (ATP binding site)
- 2Xcalmodulin light chains at neck region (regulate)
- tail region interact with plasma membrane
What is the structure of Myosin II?
- dimer
- 2 globular head regions with ATPase activity (ATP binding site)
- 2X regulatory light chains at neck region, and 2Xessential light chains
- tail region (130nm) interact with plasma membrane
What is the structure of Myosin V?
- dimer
- 2 globular head regions with ATPase activity (ATP binding site)
- light chains at neck region
- tail region interact with vesicle membrane
What are the functions of Myosin I, II, V?
Myosin I: cytoskeletal - plasma membrane interactions (formation and movement of filopodia and microvilli)
Myosin V: interaction between cytoskeleton and vesical membrane (transport)
Myosin II: muscle contraction and cytokinesis
-head region bind actin, light chains at neck region regulate head domain, coiled domains pack side by side to form a thick filament
How is formation of Myosin II bipolar filament regulated?
By myosin light chain kinase (MLCK)
- following activation mysin light chains in the neck region are phosphorylated by MLCK (elavated Ca2+)
- activates actin binding domain and causes a conformational change
- results in release of the tail and spontaenous self assembly to form a bipolar filament structure
What experimental assay can be done to detect Myosin II activation?
The sliding microfilament assay
1- myosin molecules are attached to a coverslip
2- washed with bovine serum album (protein wash) to get rid of non-adhearent protein molecules
3-Actin filaments fluoresently labelled (Rhodamin phalloidin) are added to the coverslip and bind physically with the actin molecules
4- when ATP is provided myosin II goes through contracile interaction movement, moving the labelled actin filaments which can be viewed by a fluoresence microscope
What is the process of muscle contraction?
1- Myosin heads are bound to the actin filament in the resting state
2- Influx of CA2+ binds to troponin on actin filament causes a conformational change in tropomyocin, exposing myosin binding sites
3- ATP binding to myosin II causes a conformational change in myosin, disrupting the actin binding site and releasing the myosin heads
4- ATP is hydrolysed to ADP and Pi restoring the actin binding site and causing a conformational change so that the myosin head pivots and binds to a new actin subunit
5- Pi is then released causing the ‘power stroke’ conformational change which pulls the actin filament
overlap to shorten
What is the Z disk?
Actin microfilaments are anchored to Z disk to allow myosin II to ‘pull’ and generate suffient force to move
