Topic 6 - Cytoskeleton Flashcards
1
Q
What is the function of Intermediate Filaments?
A
- mechanical strength so holding the cell together
2
Q
What is the function of Microtubules?
A
- positions the organelles in the cell
- directs INTRAcellular transport
3
Q
What is the function of Actin Filaments?
A
- sets the shape of the cells surface
- used for cellular locamotion
4
Q
Microtubules are made up of what?
A
- tubulin
5
Q
Why are cytoskeletal filaments dynamic?
A
- allows for a process such as replication to bend and shape the cell
- microtubules are involved in the mitotic spindle
- while actin form the contractile ring
6
Q
What is responsible for the polarity in a cell?
A
- cytoskeletal filaments
- Microtubules for the coordinate system for the cell by directing intracellular locomotion
7
Q
Why does the cell disassemble filaments and reassemble them instead of moving the whole structure?
A
- allows the cell structre to rearrange based on cellular needs
- cell is able to rapidly disassemble filaments and diffuse these subunits to a desired location to reassemble there
8
Q
What is the importance of protofilametns of the cytoskeleton? - compare single vs multiplexed
A
- increases the thermal stability
- a single protofilament is thermally unstable since it is easy to break a middle bond or remove pieces from the end due to one interaction
- multiplexed protofilaments are thermally more stable; breaks in the middle are now multiple bond breaks & breaking pieces of the end now involved >1 bond
9
Q
Describe the structure of Actin
A
- consist of a chain of monomers intertwined with another monomer
- has a plus and a minus end (distinct polarity)
- a RIGHT hand helix will occur every 37nm
10
Q
What is the persistence length of an actin filament?
A
- between 10-40nm where the filament is STIFF but anything beyond this has thermal fluctuations leading to bending
11
Q
What process is shared by both Actin and Microtubules?
A
- polymerization and depolymerization
- addition of subunits at both plus and minus end by growth is always faster at the plus end
- there is always a balanced rate between units added (Kon) and units removed (Koff)
12
Q
What is different about the addition of subunits between Actin and Microtubles?
A
Actin: actin-ATP (T-form) –> actin-ADP (D-form)
Microtubles: tubulin-GTP (T-form) –> tubulin-GDP (D-form)
T = monomer carrying ATP or GTP D = monomer carrying ADP or GDP
13
Q
What is the rate like for the addition of subunits?
A
- considering the plus end: K(T)on vs K(D)off – for the hydrolysis of the ATP/GTP to ADP/GDP on the subunit
- typically though the addition of subunits are faster than hydrolysis of them
14
Q
What are the 3 key stages of the Critical Concentration? (Cc)
A
- Nucleation (formation of a trimer)
- Elongation (polymer of actin)
- Stability steady (treadmilling)
15
Q
What is the Critical concentration? Cc?
A
- once the steady state is reached, it is the equilibrium concentration of the pool of unassembled subunits
- LOSS=GAIN
16
Q
What are the parameters of Cc?
A
- at [monomer] below Cc no polymerization occurs
- at [monomer] above Cc filaments assemble until monomer concentration reaches Cc again
17
Q
What are the two type of actin we get?
A
- G actin (globular) - monomer
- F actin (filament) - polymer of actin
18
Q
What two steps go hand in hand for nucleation and elongation phases?
A
- polymerization with ATP/GTP while there is hydrolysis to ADP/GDP
19
Q
Explain the nature of growth speed with hydrolysis of the filament
A
- PLUS end ADDITION of subunits via polymerization is fast and races ahead as hydrolysis lags behind
- MINUS end ADDITION of subunits via polymerization is SLOW and hydrolysis will attempt to catch up with the plus end
20
Q
What does ATP,GTP > ADP,GDP mean?
A
there are more soluble units in the T form (polymers are always in a mixture of T form and D form)
21
Q
Are T-form and D-form equally likely to be (de)polymerized?
A
- T form is more likely to be polymerized and faster at the plus end than the minus end into the filament where they are hydrolyzed into D form as stored energy
- D form depolymerizes faster than T form
22
Q
Growth at either end of the filament is dependent on two things?
A
- hydrolysis of the subunits on the filament
- concentration of the different subunits (increase in [subunits] growing end will be in T form VS decrease in [subunits] growing end will be D form and depolymerize)
23
Q
What does tread-milling refer to? and what are the two key points (goes back to Cc)
A
- when polymer gains @ plus end = polymer loses @ minus end
- the concentration of free monomer is above the Cc @ the plus end = assembly
- the concentration of monomer is below Cc @ the minus end = disassembly
- essentially both ends of the filament are exposed during steady state; there is an identical rate of net assembly at the plus end and net disassembly at the negative end
24
Q
What does dynamic instability in microtubules refer?
A
- raid growth or shrinkage may occur
25
What does "catastrophe" refer to?
- sudden switch of a growing microtubule into a rapidly shortening state
26
What does "rescue" refer to?
- occurring after catastrophe GTP-bound tubulin or ATP-bound actin can begin adding to the tip of the microtubule/actin again, providing a new cap and protecting the microtubule from shrinking
27
What is the GTP/ATP capping refer to?
- GTP-bound tubulin or ATP-bound actin is proposed to exist at the tip of the microtubule/actin, protecting it from disassembly
- sensitive to hydrolization which may cause sudden catastrophe
- capping actin at the plus end is done by CapZ while capping actin at the minus end is tropomodulin
28
What two enzyme work against each other when it comes to free actin monomers?
- THYMOSIN works by binding free actin and inhibiting PLUS END GROWTH
- PROFILIN works by binding free actin and inducing RAPID plus end growth (by adding ATP to the monomer)
- thymosin/profilin compete to bind the free monomer of actin but cannot both bind at the same time
29
What are 3 important feature of actin nucleation?
- frequently occurring at the plasma membrane (actin filaments is used to maintain plasma membrane structure)
- regulated by external signal
- regulated by the ARP complex or FORMINS
30
What role does Formins have in actin filament formation?
- a dimeric protein associated at the growing PLUS end
- captures to actin monomers
- produce straight unbranched filaments
31
What is the ARP complex?
- actin-related proteins
- an ARP complex must activate by binding an activating factor
- once ARP is active it will initiate nucleation by binding actin monomers and initiate growth in the POSITIVE PLUS END direction
- so ARP complex works from the negative MINUS END
- will produce branches at 70˚
32
What is substantially different comparing formins and ARP complex? - both work with actin though
- the ARP Complex works with the negative end
| - Formins work with the plus end
33
Explain step by step the polymerization and depolymerization of G-actin to F-actin
G-actin (polymerize) F-actin-ATP (hydrolyze) F-actin-ADP (depolymerization) G-actin-ADP (ADP/ATP exchange) G-actin-ATP
34
Bundles are made of...
| Webs are made of...
- long-straight filaments by FORMINS
| - ARP Complex
35
What are the two different types of bundles that can form? and what is the key difference?
a) contractile bundle - loose packing allows myosin-II to enter the bundle
b) parallel bundle - tight packing prevents myosin-II from entering bundle
36
Formin and ARP Complex share what characteristic and what difference?
- Formin associate with the plus end and nucleates assembly of BUNDLES
- ARP complex associates with the minus end and nucleates assembly of WEBS
37
Thymosin and Profilin compete for what?
- both compete to bind free actin monomers
- thymosin binds to prevent assembly
- profilin binds to speed up elongation via rapid growth
38
What do tropomodulin do in the cell
- prevents assembly and disassembly at the minus end
39
What are the 3 goups of motor proteins in a cell?
- myosins (actin - motor)
- kinesins (microtubule - motor)
- dyneins (microtubule - motor)
40
What are key features to these molecular proteins?
- they are unidirectional
- utilitize ATP hydrolysis through repeated cycles
- the HEAD contains the motor: recognizes the right track & direction of travel
- the TAIL determines the cargo and biological function
- motors can move organelles, RNA molecules
- function through conformational changes
41
Which direction does myosin walk to?
- the positive end of ACTIN
42
Explain what a myosin cycle looks like?
1. Myosin bound to actin filament (lacks bound ATP)
2. myosin binds ATP at its head with reduced affinity to actin so it releases it
3. the cleft closes around ATP resulting in a shape change of the myosin as ATP hydrolysis occurs simultaneously
4. this hydrolysis causes a weak bind to a new site on actin filament -- releases of the phosphate groups causes the POWER stroke (force generating change in the shape - ADP is lost)
5. at the end of cycle, myosin head is tightly locked to actin filament in rigor conformation
43
What stage is responsible for rigor mortis?
step 1 - myosin tightly locked to actin filament after the power stroke and release of ADP
- Ca2+ has not been taken up into the sarcoplasmic reticulum and thus remain bound to troponin, which loosens tropomyosins grip on the actin which allows the stuck myosin head to remain bound to the actin
44
Quickly explain the power stroke again?
- weak binding of myosin to its new site on the filament -
next the release of the phosphate causes the power stroke: force generating change in shape (so the head regains its original conformation) and myosin tightly linked to the actin filament again
45
Why do you need Ca2+ for muscle contraction? - the Ca2+ are released after a nerve signal triggers contraction action
- the free released Ca2+ will bind to the troponin which causes a conformational change in the protein tropomyosin which binds around the actin right handed helix which functions to prevent the myosin head from binding
- and exposed actin from tropomyosin allows myosin to bind to actin and initiate the muscle contraction
46
What does calmodulin have to do with smooth muscle contraction?
- free calcium from the sarcoplasmic reticulum will bind to the calmodulin protein
- and active calmodulin with bound Ca2+ will activate the active myosin light-chain kinase
- this myosin light-chain kinase will phosphorylate the myosin light-chain and turns on the actin binding site for this myosin molecule now being able to undergo the myosin cycle
47
What is the function of myosin V?
- to transport organelles along actin cables (ex. moving a mitochondrion)
- it has long lever arms
48
What is unique to myosin V compared to myosin II
- both myosins walk towards the plus end
- Myosin V moves continuously along the actin filament without letting go
- still uses the ATP hydrolysis mechanism; one arm lets go while the other swings to the front - walking - ONE UNIT ALWAYYS REMAINS ATTACHED
49
Microtubules consist of what two components? with GTP bound how?
- alpha tubulin & beta tubulin
- alpha tubulin bind GTP very tightly
- beta tubulin binds GTP LESS tight as it has an important role in filament dynamics
50
What are the structural aspects of microtubules?
- shows POLARITY: beta tubulin as the top (as the PLUS end)
- forms a tube via 13 protofilaments
- this protofilaments makes these microtubules STIFFER than actin and harder to bend (thermally more stable)
51
What does the Persistent Length refer to in microtubules?
- the length where random thermal fluctuations result in bending
52
Where does nucleation start for microtubules?
- at the MTOC centrosome: microtubule-organizing center
53
How does microtubule formation begin?
- through the negative end via gamma-tubulin (site for nucleation)
- this minus end is very stable
54
The MTOC or Centrosome consist of what components and affects the microtubules how?
- the centrosome consists of a centrosome matrix filled with 50 copies of gamma-TuRC which forms a ring complex and the site of nucleation
- centrioles are short cylinders of modified microtubles and accessory proteins
- the plus ends (beta-tubulin ends) radiate out from the centrosome
- the minus end is collected in the centrosome and are relatively stable
55
Explain the Microtubule and Organelle Positioning system?
- the centrosome forms an astro-configuration which provides a coordinate system to organize cellular components and direct movement
56
In the experiment, what occurs when a cell is cut?
- a new MTOC will emerge
57
Where does the dynamic instability emerge from for microtubules?
- this results from structural differences between growing and shrinking at the microtubule beta-tubulin end
- a GTP cap favours growth of tubulin, while a sudden change from T-form to D-form through de-polymerization is called "catastrophe" where rapid shrinking follows
- when T-form is regained a rescue will occur forming a new GTP cap
58
When will a microtubule catastrophe or rescue occur specificially?
- growing microtubules have a GTP cap
- if T-tubulin hydrolysis catches up to the polymerization of Tubulin-GTP -- de-polymerization is favoured as the GTP cap is lost and RAPID shrinking occurs
- but GTP-subunits will begin to add onto the shrinking end, if enough attached rescue will occur and a new GTP-cap emerges
59
What does the model for structural consequences of GTP refer to?
- in a protofilament, when GTP hydrolysis occurs it weakens the wall of tubulin by changing their conformation - meaning it will be harder to pack new tubulin into the wall here
60
When a microtubule catastrophe occurs what protein is involved in the depolumerization?
- Kinesin-13 will open up the microtubules
61
What controls microtublue dynamics?
- with more than 100 microtubule binding proteins
| - the plus end binding proteins control microtubule dynamics - growth and shrinkage
62
What protein is involved in microtubule stabilization?
- XMAP215 stabilizes the growing end of the microtubule by increasing the growth rate and binding tubulin dimers
63
Kinesins are what sort of protein with what function?
- one of two microtubule motor proteins
- proteins will walk towards the plus end
- presence of two microtubules, will walk to the plus end, but the absence of a microtubule it will walk to the minus end
64
What is different about kinesin walking compared to myosin?
- in myosin when no ADP/ATP/Pi is bound to the head group, myosin is tightly bound to actin (rigor mortis)
- in kinesin however, when ATP is bound it binds TIGHTLY to microtubule surface while when ADP bound it is LOOSELY bound to microtubule surface
65
Explain how kinesin performs its "walking"
- the leading head has ADP bound and loosely associates to the microtubules
- the lagging head has ATP bound and tightly associates to the microtutuble
- when ATP hydrolysis (ATP-->ADP+Pi) occurs to the lagging strand it loosens its binding
- in the leading strand, the ADP is exchanged for an ATP thus binding tightly to microtutubles and moves the lagging head forward
- thus the new shape again has ATP bound lagging head with an ADP bound leading head
66
What is different about Dyneins?
- structurally different from myosin and kinesins
- move towards the microtubule minus end
- two different types: cytoplasmic dyneins & axonemal dyneins
67
Key characteristics of the two different types of dyneins?
- Cytoplasmic Dyneins: vesicular traffic and localizing the golgi (homodimer)
- Axonemal Dyneins: beating cilia and flagella (heterodimer & heterotrimer)
68
What is the structure of the dyneins?
- six AAA domains
- stalk is long and anti-parallel
- a ring rotates and releases ADP + Pi
- tail attaches to cargo
69
What feature does Dyneins share with Myosin?
- without ATP they are tightly bound to microtubules
- tight binding released in presence of ATP
- ADP + Pi release cause a conformational change causing a power stroke
70
What are motile cilia and flagella made from?
- from microtubules and dyneins
71
What are the differences between eukaryotic and prokaryotic flagella?
- Eukaryotic flagella are more complex and larger structure
- eukaryotic flagella made up of tubulin while prokaryotes made up of protein flagellin
- eukaryotic flagella have a bending movement powered by ATP
- eukaryotic flagella have a rotary movement driven by proton gradients
72
What sort of structure do intermediate filaments take?
- alpha helical monomer
- these monomers will form a dimer by coiling with another monomer
- a staggered tetramer forms from two coiled dimers
- 8 tetramers associate laterally to form a growing filament
73
What makes intermediate filaments so unique? - 3 examples?
- their diversity; different tissues have intermediate filaments unique to them
- main role is to provide mechanical strength
- keratin: epithelial cells
- desmin: muscle cells
- neurofilaments: nerve axon
74
Do bacterial cells have a cytoskeleton?
Yes; protein FtsZ, Mreb, CreS
- the FtsZ is a tubulin homolog (forming a ring during cell division and contains filaments)
- Actin homologs include: plasmid segregation (ParM); while MreB determine cell shape
75
Cell behavior is dependent on the coordination of... (3)
- dynamic assembly/disassembly of cytoskeletal polymers
- regulation of structure by associated proteins
- actions of motor proteins
76
What are the three main cell movements (all driven by actin filaments)
- Filo(podia)
- Lamelli(podia) -- flattened extension of the cell, by how it moves over or sticks to a surface
- Pseudo(podia) -- temporary protrusion
77
What is the role of cofilin with actin filaments?
- binds ADP-actin filaments (D-form), and accelerates disassembly (destabilizing the polymer) at the negative end
- ATP (T-form is resistant to depolymerization by cofilin)
- with age though, the actin filament will become ADP form (D-form) and fall susceptible to cofilin (this drives the net direction of lamellipodia)
78
Are the actin filaments moving in a moving lamellipodia
YES; lamellipodia crawl forward, while the actin filament remains stationary
- since actin filaments are oriented towards the positive end, negative end is attached to the sides of other actin filaments via ARP complexes
- cofilin cycle responsible for the net directional movement in lamellipodia
79
What are Rho proteins? particularly: Cdc42, Rac, Rho?
- Rho: activation results in stress to fibres
- Rac: activation leads to lamellipodia
Cdc42: activation leads to filopodia(microspikes)
- Rho proteins function like switches to control cell switches (a monomeric GTPases)
80
What does active monomeric GTPase (Rho-GTP) do within a cell?
- activates formins (promoting actin bundles)
- activates ProtK = inhibitor of cofilin (thus stabilizing the filaments)
- inhibits phosphotase which acts on myosin chains (increasing myosin motor activity)
81
What is the role of Rac-GTP?
- activates crosslinking (ARP complexes)
- inhibits contractile myosin (decreases myosin activity)
- stabilizes lamellipodia
82
Explain the extracellular signalling occurring in lamellipodia formation?
- Chemotaxis signalling move bacteria, these external signals are diffusible and non-diffusible
- there will be local stimulation of polymerization of actin near the receptor; this occurs via activation of Rac GTPase via G-protein and PIP3 -- Rac-GTPase activate ARP complex and lamellipodial protrusion
83
Explain the extracellular signalling occurring in actin-myosin contraction?
- the external signals diffuse further via G12/13 which will activate the Rho pathways
- actin-myosin cause contraction
84
Why does it make sense to activate two signaling pathways that inhibit each other?
- these two pathways are mutually antagonistic
- initially actin polymerization at the plus end protrudes the lamellipodium
- the cells cytoskeleton must first attach itself to the lamellipodium and the plus end before initiating contraction via the actin-myosin
