Actin Flashcards
1
Q
What are three types of cytoskeleton?
A
- Microfilaments (Actin)- usually in the cortical area
- Microtubules (tubulin?)
- Intermediate Filaments
2
Q
what are functions of cytoskeleton?
A
cell shape, intracellular and whole cell movlemtns, for roganizling cell contents, and for integrating cells with other cells though specialized adhesions
3
Q
What are sizes of each cytoskelton?
A
Microfilaments - Actin0 7-9 nm
Microtubules are 25 nm
-alpha and beta tubulin proteins
Intermediate filaments - 10-11 nm
IF proteins
4
Q
what is the primary “mover and shaker” of cellular behaavior?
A
neutrophil in blood moving towards bacteria
5
Q
what does it mean to be dyanmic
A
assembly adn disassembly
6
Q
What is another name for atin skeleton?
A
Microfilaments
7
Q
What part of cell is actin filametns most concentrated
A
Cortical region near the plasma membrane
8
Q
What percent of the total protein in NON MUSCLE cells are actin?
A
5-10%
9
Q
What % of the total protein in skeletal muscle are actin?
A
> 20%
10
Q
How many acitn genes are in humans?
A
6>
encode different isoforms have minor differences in seuqence
11
Q
Where are ALPHA-ACTIN present?
A
ONLY in MUSCLES
12
Q
Where are beta and gamma actin presetn?
A
Together in no-msucle cells
13
Q
Actin drives activity of entire cell? T/f
A
True!!! Whoel cell
14
Q
what other specialized structures can you find actin>
A
lamellipodia, stress fibers, microvilli, others
15
Q
what is actin called in its unassembled form?
A
G-actin (“the OG actin”
16
Q
How big is actin
A
40-42 kilodalton globular protein
17
Q
How many actin monomers are present/turn?
A
2 actin monomers /turn
18
Q
where does actin monomers bind ATP?
What direction does the ……..bind? (neg or pos)?
A
Region of molecules called ATP-binding cleft
All actin subunits are going in same dirction
ATP binding cleft points to the (-) end
19
Q
What does nucletotide binding (ATP) do to the molecule?
A
affects conformation of actin moleucel and makes it more assembly-competent
20
Q
What is actin called once it’s polyerized
A
Filamentous or F-actin= MICROFILAMENT form
21
Q
what conditions favor actin microfilament assembly?
A
- sufficient conecntration of subunits
- Physiological salt concentration
- ATP
- Calcium
22
Q
Does actin form spontaneously?
A
YES!
23
Q
The assembly of actin into MF is accompanied by delayed hydrolysis of ……
A
ATP! …atp hydrolysis is delayed with seebly of actin
24
Q
Is energy required for assembly?
A
No!!! Assembly occurs in teh presence of non-hydrolyzing ATP Analogs
25
What is problem for cells that favor actin assembly?
TOO MUCH ACTIN under good conditions!
Keeping all actin fivers from assebmly into Microfilaments
restricting.directing assembly of MF to speciic cell locations
26
What are three stages of actin polymerization?
1. nucleation (seeding)
2. Elongation
3. Steady state (equilibirum)
27
what Happens at slow nucleation/seeding stage? What is the "seed"?
initial lag pahse
1st few monomers to form TRIMERS OR SHORT OLIGOMERS OF ACTIN OLIGOMERS which are the "nuclei seed to contineu with elongation?
28
What is the critical concentration
the concentration of actin subunits at steady state equilibirium (build/diassasemle rate equilibirum)
29
Do cells use special proteins to help nucleate near filaments at specific sites?
Yes!
30
is the Cc changed with the addtion of a nuclei?
NO! not changed! just decreases lag phase
31
Is polarity more exaggerated for actin filaments or for microtubule filaments?
Actin filaments have a much more exaggerated polarity !
At cell
32
How fast is growth during cellular conditions for actin? plus end vs minus end
it is 10x fster at the plus end than the minus end!!!!
33
which end (plus or minus) is more dynamic?
plus end
34
how much faster does plus end grow at cellular conditions?
10x`
35
Can actin catalyze the hydrolysis of ATP?
Yes, for the fee subunits, this is extemely slow
This is delayed after actin subunits are assembled into filaments
36
Are soluble units (not polymer) T form or D form
T form !
37
Are polymers T form or D form?
It is a mixture of T form and D form!
38
Compare Critical concentration of T form Vs D form
Cc T form (+ end) < Cc of D form (- end)
39
Can subunits assemble when ATP bound (T form) or not bound (D for)
Bound!! T form
40
Where is the Energy from high energy phosphate stored?
In the POLYMER LATTICE.
contributes to different equilbiruim concentration (critical concentations)
41
Difference between plus end and minus end with assembly
As filament grows, plus end is more addtion is fastre and ATP hyrdolysis lags behind assembly, leaving cap of ATP subunits at plus end
CAP of ATP suunits at plus end b/c ATP hydrolysis lags
42
What is T forma nd what is D form ?
T form = subunits with bound ATP
D form- subunits without bond ATP (ADP is bound)--hydrolysis caught up
43
What is Critiical concentraiton?
is [x] is > Cc --> subunits are added (growing)
If [x] subunits removed (shortening)
44
What concentration does Treadmilling occur in Actin?
Between CC of plus and minus end
Cc(T) plus end < treadmilling < Cc (D) minus end
45
What concentration does net assembly occur
at high monomer concentration (concentrations above Cc D)
46
What direction (+) --> (-) or (-) -->(+) does newly added subunits move?
From positive (added side) to minus (removed)
47
waht end/part of cell can you find actin nucleoation / (+) end location
Positive end Near the PM of the cell
addition of actin occursat plus ends and newly added subunts treadmill their way deeper into the cells central regions where negative sings are located
48
What drugs affect actin filaments? (4)
1. Phalloidin
2. Cytochalasin
3. Swinholide
4. Latrunculin
49
What does Phalloidin do to actin?
Binds and STABILIZES FILAMENTS
50
What does Cytochalsasin
caps filament plus ends
51
What does Swinholid e do?
Severes filaments
52
Latrunculin
binds subunits and prevents their polymerization
53
What poisons are contained in Amanita Phalloida (Death Cap Muschroom)
1. Phalloidin -
binds f-actin with high affinity
-inhibits its depolymerization and disrupts MF function
2. Amanita-
attacks RNA Polymerase II 0 resulting in liver and other organ failure
54
What does Phalloin do?
High affinity to f-actin--> prevents depolymeriastion
55
What does Amanita do?
Attacks RNA polymeraase II --> liver and organ failure
56
What are examples of non-motile actin filametns?
Microvilli and stress fibers (holds cells down)
57
What are examples of Motile MF Assemblies
1. Filopodia and Lammeliopoda
| 2. Contractile ring (cytoskeleton during anaphase? )
58
what are actin binding proteins?
organize ceullular assembles of actin filaments
59
What is the function of cutting/severing proteins?
Control lengths of Actin Filaments
60
What is an example of a cutting/severing protein?
Gelsoilin- which is Ca Activated
61
What is the function of Gelsoilin
Activataed by ca2+ --> attcks F-actin --> cuttign it into pieces
62
What part of actin can gelsolin bind
2 actin bidning sites located alont eh SIDE and END of an actin filament
63
What is the result of the fragmentation of actin by GELSOLIN
Does Gelsolin require Ca?
more "oslated" or fluid-like consistency of cytoplasm
Yes, requires ca
64
what are two (loose) classes of actin cross-linking proteins
1. Gel forming proteins (larger in size)
| 2. bundling proteis
65
What is an example of a cross-linking protein
FILAMIN- acts as a dimer to crosslink actin fibers
66
What is FILAMIN
dimer that crosslinks actin (actin cross-link protein)
67
What kind of array does "bundling" type of actin form?
Parllel arrays of actin microfilaments
Can be LOOSE! Can be TIGHT!
68
Example of loose bundle protein
Actin Filament + ALPHA- ACTININ
allows Myosin II to enter bundle (contractile bundle)
69
Example of Tight Bundle
- Actin Filaments + FIMBRIN!
Parallele bundle that is TIGHT! prevents Myosin II from entering bundle
70
What type of proiten is found in core of Microvilli
FIMBRIN!!!!
71
Are microvilli motile/
NO!
72
What is the core of the Microvilli made from
Core of tightly bundld actin filaments (Dense actin filaments)
73
What part of microvilli cell has positive end polarity?
(+) ends closest to tip of microbilli
74
What is at the base of the microfilament core?
Terminal web@@@
contains meshwok of filaments that contains rootlets of the actin cores perpendicular actin MF, IF, and other proteins
75
What is Celiac Disease? Cause?
Disease that damanges the SI and interferes with absorption of nutrients from food
Cause: Damaged microvilli in SI from gluten ingestion
76
Cause of CD (Celiac Disease)
Peopel with CD cannot tolearte protein GLUTEN
Auto antibodies to actin smetimes found in CD patients
77
Presentation of microvilli in CD pateints
Microvvilar surface (brush border) is much thinner --> decrease abosptive SA
78
Why do you need adapter proteins?
To link actin network to PM (actin doesnt bind to any membrane protein)
79
membrane ADapter proteins for platelet?
FILAMIN
Filamin links MF to Glycoprotein complex in membrane
80
Filamin vs Fimbrin
Filamin- adapter protein for platelets
Fimbrin- cross linking protein taht result in tight bundles of actin - Fimbrin found in core of microvillus
81
What is membrane Adapter protein in Muscle Cell?
DYSTRPHIN-
attracks actin filament to integral glycoprotein complexes
82
What doe s defect in Dystrophin Gene Cause?
Duchemme's Muscle Dystrophy
Fatal
83
proteins in apical membrane of various epithelial cells are linked to actin MF by what?
ERM family of proteisn
(Erzin, Radizin Moesin)
direclty or through intermediate proteins such as EBP50 or CFTR prtoein (Cystic fibrosis TM conductance regulator protein
84
What is the major protein that provides structural basis for cortical cytoskeleton in RBCs
1. Actin Binding Proteins SPECTRIN (dimer)
undertneath PM
Takes a lot of abuse
85
What does the RBC elaborate cortical cystoskeleton consist f?
Spectrin
Actin
Adaptors
86
What is the structure of Spectrin
Long Alpha and Beta Chains associated laterally to form DIMERS then Tetramers with actin binding domains at both sides
87
What do ends of spectrin TETRAMERS bind to?
Short Actin Filaments
88
What is protein ANKYRIN
Ankyrin binds to spectrin and TM proteinBadne 3
89
What are Protein 4.1 binds to ?
Protein 4.1 binds to spectrin-actin jucntions and to Glycophorin, an abundant TM protein
90
What is the problem with solutlbe monomer concentrations of actin?
High in cells
| Problem keepign them from polymerizing
91
Solution to polyermizations
Seqeunstering monomeric actin using special actin binding proteins
Thymosin and Pofillin
92
what are the most abundant sequestering proteins?
Thymosin- locks actin monomers cannot assempt
93
What is profillin?
Competes with thymosin for actin monomer
Prifllin actin dimers can readilya dd to actin filametns
Adds to face of actin OPPSOITE the ATP bnidng cleft (which is on teh minus end)
Cannot bind to minus end of actin ,but easily adds to positive end
94
How to you "activate" profillin ?
By Phosphorylation and inositol phospholipid binding (IP3)
95
What is result of locally activating profilllin
movment of actin from the seqeunstered thymosin pool into an ASSEMBLY Pool
Actin moves from sequenstered pool --> Assembly pool
96
Fucntion of Formin Proteins?
large family of Actin Binding PRoteins taht fomrs Dimeric Complex--> NUCLEATES new actin filaments --> elongates
97
hat are ends of Formin protein associated with?
Rapidly growing postive ends of actin filaments as they elongate
98
What are "whiskers"
contain binding sites for profillin or the Profiln- Actin Dimers (Right side)
Serve as "stagin area" to inrease rate of actin filametn elongation
99
Formin protein fucntion? What doe formin proteins typically nucleate?
Nucleats growth of striaght, unbranched filaments whih link into parallel bundles (unlike ARP mediated actin webs)
100
What is an ARP?
Actin Related Protein complex
101
What is the structure and functino of ARP
ARP 2 and ARP 3 have a strcuture similar to actin monomer but unable to form filaments on their own (not spontaneous)
102
Can ARP form filaments on their own?
NO
103
What happens when they are activated
Forms a complex that mimics the plus end of MF
simulates NUCLEATIOn and growt of new actin filaments
104
What is the result and end product /structure of ARP activation
Bc of unique binding of ARP complexes to the sides of existing filaments wit a particular configuartion, a web of actin branches form from existing filaments
105
Difference betwen the nucleation caused by Formin proteins and the Nucleation caused by ARP proteins
Formin protein nucleation- strainght unbranched filaments (actin filaments can sponaneously grow)
ARP proteins- similar sructure to actin monomer, nuclates to form a WEB of actin BRANCHING from existing filaments
70 degre branches
106
Where is nucleation by ARP mediated at?
Leading Edge
107
what angle are newly nucleated actin filmants attached
70 degrees to side of pre existing filament
108
What is cofillin?
Destabilizes Actin Filament (it's an actin filaminet binding protein)
109
What makes Cofillin unusual?
It binds to both filament and globular forms of actin
110
What form of actino for Cofillin prefer?
ADP containign actin filaments (older filaments)
111
What does binding to COfillin do?
Makes it easier for ADP actin to dissociate from minus end
112
Overall
Enables actin filaemnt entowrk as a whole to move forward, pushing out leading edge of a moving cell
113
How does cell walk i.e. lamellooodeim
1. extention/protrusion (actin polymerization)
2. movement of unpolymerized actin--> forms translaocation
3. Detarchmentand contraction of trailing bak end
4. Afoward movment (actin myosin interaction)
