Module 4 Flashcards
(56 cards)
1
Q
What are the roles of the cytoskeleton?
A
- adopts a variety of shapes
- carries out coordinated direct movements
- divide by binary fission
- organize its intracellular space
2
Q
What is the structure of the cytoskeleton?
A
- cytoskeleton is a number of proteins that polymerize into filaments
- dynamic and complex
- continuously changing according to the needs of the cell
- made up of microfilaments, microtubules, and intermediate filaments
- distribution and organization of the filaments polarizes different types of cells
3
Q
What are actin filaments?
A
- a type of microfilament
- polymers of actin monomers
- forms a helical structure
- flexible and dynamic
- can lengthen and shorten
- 7-9 nanometers in diameter
- visible on transmission EM
4
Q
Where are actin filaments found?
A
- microvilli
- cell cortex
- Adherens belt
- filopodia
- lamellipodium/leading edge
- stress fibers
- contractile ring
5
Q
What is the role of actin in the cell cortex?
A
- protects the cell
- contributes to the structural integrity of the tissues
6
Q
What is the role of actin in Adherens belt?
A
- structural integrity
- plays a role in development when the epithelium is changing structure to move tubules
7
Q
What is the role of actin in motile cells?
A
- actin is in filopodia and lamellipodia
- actin is also found in the cell cortex
- helps with movement and structural integrity
8
Q
What is the role of actin in stress fibers?
A
assists in cell adhesion
9
Q
What is the role of actin in the contractile ring?
A
assists in cell division
10
Q
What are intermediate filaments?
A
- intermediate size to all 3 filaments
- 10 nm in diameter
- made up of polymers of intermediate proteins
- forms rope-like structures
- provides structural support and helps maintain shape
11
Q
Where are intermediate filaments found?
A
- in the tight junctions between epithelial cells
- help with cell-cell interactions
- also surround the nucleus to offer protection and support
12
Q
What are microtubules?
A
- polymers of tubulin proteins
- the largest of the 3 filaments
- 25 nm in diameter
- hollow and cylindrical in shape
- very rigid
- long and stiff
13
Q
Where are microtubules found?
A
usually emanate from a single point in the cell known as the microtubule organizing center (ex: centrosome)
14
Q
Which two filaments have a similar pattern of distribution in the cell?
A
- intermediate filaments and microtubules
- microfilaments have a more dynamic, unique structure
15
Q
What is the monomer of actin called?
A
globular actin (G-actin)
16
Q
What is the polymer of actin called?
A
filamentous actin (F-actin)
17
Q
Which end of actin are monomers added to?
A
the plus end (fast growing end)
18
Q
What is the difference between the plus and minus end of actin?
A
- the plus end (barbed end) is the fast growing end where monomers are added
- the minus end (pointed end) grows more slowly because monomers are usually not added here
19
Q
What are the steps of actin polymerization?
A
- Nucleation: A group of G-actin monomers comes together and forms a trimer polymer that is referred to as a seed/nucleus. This is the slowest step (rate-limiting step).
- Elongation: In vitro, monomers are added to both the plus end and the minus end to get rapid growth of the filament. In vivo, monomers are usually only added to the plus end.
- Steady state: At a certain concentration of G-actin concentration known as the critical concentration, the rate of monomers being added to the filament is equal to the rate of monomers coming off of the filament. There is no net growth during this stage.
20
Q
What will happen if the concentration of G-actin is higher than the critical concentration?
A
G-actin will polymerize into F-actin until the critical concentration is reached
21
Q
What will happen if the concentration of G-actin is less than the critical concentration?
A
F-actin will depolymerize into G-actin until the critical concentration is reached
22
Q
What phase of actin polymerization represents the lag phase?
A
- nucleation
- takes time for the monomers to come together to form a nucleus
- represented by a flatter line on the graph
23
Q
Why does G-actin bind ATP?
A
- in order to become polymerized, the G-actin monomer needs to bind ATP
- as the filament ages, the older monomers of the filament will have their ATP hydrolyzed to ADP
- when ATP is hydrolyzed to ADP, this promotes depolymerization of these older monomers
- these monomers can then be recharged with ATP
- if ATP can not be hydrolyzed to ADP, then actin will permanently stay in the polymerized form
24
Q
Why are G-actin monomers added to the plus end and taken away from the minus end?
A
- the critical concentration at the minus end is different from the critical concentration at the plus end
- the critical concentration is substantially higher at the minus end than it is at the plus end
- therefore, you typically see polymerization at the plus end and depolymerization at the minus end
25
What is the treadmilling effect?
- the apparent movement of monomers through the filament from the plus end to the minus end
- continuous addition at the plus end and subtraction from the minus end
- you can track the monomers with a GFP label, which will show them appearing to move on a "treadmill"
- the actin filaments can polymerize in certain parts of the cell in a treadmill fashion to create the forces necessary to extend the membrane in a certain direction
26
What is the rate of addition of G-actin at the plus end?
12 micromolar/second
27
What is the rate of addition of G-actin at the minus end?
1.3 micromolar/second
28
What is the rate of depolymerization at the plus end?
1.4 micromolar/second
29
What is the rate of depolymerization at the minus end?
0.8 micromolar/second
30
What is the implication of the association and dissociation rates for actin dynamics?
- the critical concentration at the plus end is 0.12 micromolar (1.4/12)
- the critical concentration at the minus end is 0.60 micromolar (0.8/1.3)
- at a G actin concentration higher than the critical concentration, association will occur
- at a G actin concentration lower than the critical concentration, dissociation will occur
31
What is cytochalasin and what does it do?
- a metabolite from fungi
- causes the depolymerization of actin
- binds to the plus ends of the filaments to prevent the addition of monomers to this end
- eventually all of the monomers will come off of the plus end
- if cells are treated with cytochalasin, shortly all of the filamentous actin will become depolymerized
32
What is latrunculin and what does it do?
- a metabolite from a species of sponge
- causes depolymerization of actin
- binds to actin monomers and sequesters them to prevent them from being added
- if you add enough, you can bind all the monomers in the cell
- no longer get addition at the plus end
- still get loss at the minus end
- eventually get depolymerization of the filament
33
What is phalloidin and what does it do?
- a toxin from a fungus
- aka Death cap mushroom
- very lethal
- stabilizes actin in the cell
- prevents actin from growing and shrinking
- no longer have loss at the minus end
- may prevent addition to the plus end
- fixing and freezing the actin filaments in time
- typically attacks liver cells
- Silibinin is a drug that comes from the milk thistle plant that is a treatment (not yet FDA approved)
34
Which proteins regulate the polymerization of actin?
profilin (positive regulator) and thymosin beta4 (negative regulator)
35
Which proteins regulate the length of actin?
cofilin and gelsolin
36
Which protein regulate the nucleation and branching of actin?
Arp2/3 complex
37
Which protein regulates the cross-linking of actin?
filamin
38
Which protein is a motor protein that moves along actin?
myosin
38
Which protein is a motor protein that moves along actin?
myosin
39
Which proteins regulate the stability and capping of actin?
CapZ and tropomodulin
40
Which protein regulates the organization of actin filaments?
nebulin (muscle cells)
41
What does profilin do?
- responsible for charging G-actin monomers with ATP and then cofilin
- can increase the speed of treadmilling
- binds to ADP actin monomers on the opposite side of the protein
- when profilin binds to the ADP actin monomers, they change shape so that the cleft holding the nucleotide opens up a bit and you get diffusion of ADP out of the cleft
- ATP then diffuses into the cleft
- second function: prevents the interaction of the ATP bound G-actin monomer with the minus end
42
What does cofilin do?
- binds to portions of the filament where concentrations of ADP actin is high (older parts of the filament)
- changes the shape of these filaments slightly so that they become destabilized
- filament becomes severed and large pieces come off of the minus end
- this opens up the minus end for depolymerization to occur
- increased treadmilling in these filaments
43
What does thymosin beta 4 do?
- can bind to ATP G-actin to prevent its incorporation to the plus end of actin
- thymosin beta 4 bound G-actin monomers and free G-actin monomers are in dynamic equilibrium
- if G-actin concentration goes down, some G-actin monomers will be released from thymosin beta 4
44
What are the steps of actin motility?
1. Step 0: trailing edge must adhere the substrate before lamellipodium can be extended
2. Extension: stretching or outgrowth of the leading edge of the cell to form a structure known as the lamellipodium which is a membrane structure that resembles a foot (polymerization of actin important here)
3. Adhesion: the lamellipodium touches the surrounding extracellular matrix and the extracellular matrix components
4. Translocation: forward movement of the cell body
5. Deadhesion: the lagging edge at the rear of the cell is released from the matrix
45
What does the Arp2/3 complex do?
- made up of 7 proteins
- 2 important proteins are Arp 2 and 3
- generates the force to drive the leading edge of the cell forward
- nucleation promoting factor (NPF) can bind one actin monomer
- 2 NPF-actin complexes bind the Arp2/3 complex
- this interaction induces a change of Arp2/3 complex and it attains a high affinity for binding to the side of an actin filament
- the actin monomer can then initiate or nucleate actin formation in a different direction
- new branch is 70 degrees from the plus end
46
What are the members of the myosin family?
myosin I, II, and V
47
Myosin I
- 1 single heavy chain
| - 3 light chains (calmodulin)
48
Myosin II
- most abundant myosin
- muscle myosin
- 2 heavy chains that are coiled-coil structures for 2 alpha helices
- 2 heavy head domains that contain actin binding sites
- 4 light chains
49
Myosin V
- two heavy chains
| - many light chains associated with myosin 5 are calcium binding proteins such as calmodulin
50
How does a myosin protein move along an actin filament?
1. Myosin head begins in the rigor conformation where it is not bound to ATP and will bind tightly to the actin filament. Myosin is also bound to cargo.
2. Upon binding to ATP, you get a conformational change in the myosin head that releases its hold on actin.
3. The bound ATP is hydrolyzed to ADP and Pi by the ATPase activity of the myosin head. The myosin head then binds to actin again and is moved toward the plus end.
4. Inorganic phosphate and ADP are released so that the myosin returns to the rigor position. The cargo is moved towards the plus end.
* Calcium is required
* Myosin VI moves towards the - end of actin while all other myosins move toward the plus end
51
What is the role of Myosin VI?
- only myosin to move toward the minus end
- plays a role in endocytosis
- binds to endosomal vesicles and moves them toward the center of the cell
- plus end of actin is toward the plasma membrane and minus end is pointed toward the center of the cytosol
52
How are myosin neck length and distance traveled in one step/velocity related?
distance traveled in one step and velocity are directly proportional to neck length
53
What is the step size of myosin V?
72 nanometers
54
What do CapZ and tropomodulin do?
- CapZ binds to the plus end of the actin filament
- tropomodulin binds to the minus end of the actin filament
- tropomodulin keeps monomers from being lost from the minus end
- both add stability to the sarcomere structure
55
What do titin and nebulin do?
- titin binds myosin and acts like a spring
- titin connects myosin and the Z disk
- nebulin binds actin and adds stability
- both maintain the organization of the sarcomere
