lecture 8 Flashcards
1
Q
why are there differences in actin in a cell vs. test tube
A
due to presence of actin binding proteins that regulate cytoskeletal form and function
2
Q
what is first type of actin binding proteins
A
control filament assembly
3
Q
what is second class of actin binding proteins
A
responsible for nucleating actin filaments/cytoskeleton
4
Q
third class of actin proteins
A
shape structure of those filaments [arranges actin filaments]
5
Q
what proteins in first class
A
thymosin, profilin, cofilin
6
Q
what proteins in second class
A
formin, arp 2/3
7
Q
what proteins in third class
A
fimbrin, alpha actinin, filamin
8
Q
what do proteins that control filament assembly do
A
control the rxn of where monomers are being added or removed from actin filaments
9
Q
what does thymosin do
A
binds to actin monomers and prevents them from being added to actin filaments
10
Q
what does profilin do
A
opposite of thymosin; binds actin monomer and puts it on actin filaments hella fast (so it happens much faster than in test tube)
11
Q
what does cofilin do
A
binds to existing actin filaments & breaks them apart into component pieces
12
Q
why is cofilin important
A
limited # of monomers in cell, so it recycles old filaments into monomers so they can be used in new actin structures
13
Q
what do actin binding proteins responsible for nucleating actin cytoskeleton do
A
kinda like cofactors/enzymes that catalyze nucleation step
14
Q
why is nucleation faster in cell vs test tube
A
b/c of proteins that provide as scaffold to bring oligomer together in right place & right time –> overcome penalty of time it takes to seed a new filament
15
Q
what do these 2nd class of proteins generate
A
2 distinct actin networks
16
Q
what is formin
A
nucleates & facilitates polymerization of long, straight actin filaments
17
Q
what is arp 2/3
A
branched, straight actin filaments
18
Q
what does arp 2/3 do
A
does a branch coming off of a pre-existing filament –> enormous branched actin netowrk which forms lamellipodia
19
Q
what is lamellipodia
A
leading edge of migrating cells
20
Q
what kinds of filaments can the third class of protein make
A
filaments that are branched, tightly, or loosely bundled
21
Q
what do different arrangements allow actin to do
A
function in specific ways in diff parts of cell
22
Q
what are proteins that arrange actin filaments
A
fimbrin, alpha actinin, filamin
23
Q
what is fimbrin
A
short cross linker; takes those long straight actin filaments and bundles them together into a very tightly woven bundled actin fiber
24
Q
what is alpha actinin
A
loosely bundled filament, essential for helping myosin to fit in actin fibers to create contractile machinery (actomyosin stress fibers)
25
what is force generating aspect of cytoskeleton
alpha actinin
26
what is filamin
holds actin filaments together; flexible, dimer
27
describe filamin
can bend, stretch, adopt angles
28
where is filamin found
underneath plasma memrbane of cells in a cortex
29
what does filamin provide for cells
little more resilience so they don't burst upon mechanical stress
30
what powers cell movement
myosin II contractility
31
what does profilin do
accelerates actin monomer addition to plus end
32
what does thymosin do
blocks actin monomer addition to plus end
33
what does monomer availability control
actin filament assembly
34
how does thymosin work
floating around in cytoplasm, bumps into actin monomer & binds it --> blocks addition to plus end
35
what happens when actin is bound to thymosin
cannot be added to growing actin filament
36
when is thymosin active
all the time; breaks are always on
37
what is the "accelerator" that overcomes this tendency
profilin
38
what does profilin do basically
opposite of thymosin; when it binds on actin monomer it's attaching it to an actin filament immediately
39
what is profilin like
an enzyme that catalyzes rxn
40
is profilin regulated
yes; once signal activates it, it overcomes thymosin block allowing actin filaments to polymerize
41
how does thymosin-profilin work
dynamic EQ; even tho thymosin is always present and binds actin monomers, sometimes it releases it
42
what happens when thymosin releases actin monomers
if no profilin, thymosin just grabs free actin monomer again
43
what happens if there is profilin (regulation)
whenever thymosin gives up free actin monomer, profilin grabs it + puts it on actin filament
44
what dictates whether filament grows or not
thymosin keeps doing its thing; profilin is what dictates it (if profilin is activated, free actin monomers are grabbed & directed to polymerize)
45
what one of the actin nucleating proteins that overcomes natural lag phase
arp 2/3
46
describe arp 2/3 and actin
diff gene, separate proteins but very similar structure
47
what are arp 2 and arp 3
2 of the 3 necessary monomers that nucleate an actin filament
48
what is arp 2/3 like
it's like a pre-formed dimer, shortcuts lag of nucleation by holding them in a complex
49
what does arp 2/3 need
another monomer to come along and overcome lag phase
50
can arp 2/3 be active/inactive
yes; signaling pathways can activate it
51
describe inactive arp2/3
held askew, not near each other
52
describe active arp2/3
conformational change lines them up in precise orientation
53
what happens when actin monomer is w/ active arp 2/3
rapid growth; it forms the stable oligomer that leads to rapid growth of actin filaments
54
what's the 3rd part of this arp 2 /3 complex
third actin monomer to come in to get stable seed for filament growth
55
what is NPF
signaling protein that has been activated by upstream signaling cascade that is now responsible for activating arp 2/3
56
what does arp 2/3 do
accelerate polymerization & forms branched actin filaments/networks
57
what does arp2/3 bind to most of the time
binds to a pre-existing filament
58
describe the formation of filament w/ arp 2/3
all of the previous steps happen (conformational change, actin monomer joins, fuels further growth, etc.), just anchored to a pre-existing filament when active
59
what angle is actin branch at relative to mother filament
70*
60
describe the iterative process of arp2/3
enormous branched (complex) network thru multiple copies of arp2/3 activated, forms an initial branch, then another, then another etc.
61
what does this highly branched network allow
forms a flat fan-shaped structure, allows it to push membranes forward as actin filaments polymerize
62
what is fundamental machinery that drives lamellipodia based protrusion
arp2/3
63
where are lamellipodia
formed at leading edge of cells
64
what does arp2/3 allow
instead of needing 3 monomers to come together you only need one
65
describe orientation of inactivated arp2/3
open (not near each other)
66
describe orientation of activated arp2/3
closed; conformational change --> near each other
67
where does arp2/3 complex dock onto
side of pre-existing mother filament
68
where do new active monomers join
plus end
69
what do lamellipodia drive
cell motility & junction formation
70
what does polymerization of branched actin do
push leading edge forward to drive cell movement
71
where do we see densely branched actin network
at leading edge of migrating cells in a structure called lamellipodia
72
what is job of lamellipodia
to push leading edge forward
73
what do filaments do as they grow
convert chemical E to physical force to push plasma membrane in drxn of cell movement
74
what does arp2/3 do thru its branched network forming abilities
helps form cell-cell adhesions
75
where is another place you see branched actin network created by arp 2/3
junction b/w two epithelial cells (no lamellipodia)
76
what happens to heart development if embryos treated w/ CK-666 that prevents lamellipodia formation thru inhibiting arp2/3 activity
cardiac cells would differentiate, but can't move or migrate to where heart is gonna form (cuz no lamellipodia) --> profound defect
77
what does no arp2/3 activity mean
no lamellipodia formation --> no migration (which is essential for development)
78
what is other actin nucleating protein
formin
79
what does formin do
accelerate polymerization (overcomes RDS of nucleation)
80
what does formin generate
straight filaments
81
describe molecular mechanism of formins
surfs growing plus end /(instead of sitting at minus end of filament and allowing it to grow a firm foundation)
82
what do formins continuously do
add actin monomers to plus end of growing actin filaments to generate long filaments that can be bundled into stress fibers
83
describe formin structure
dimer
84
what does dimer do w/ addition of new monomer
shifts; walks up plus end as new monomers are added
85
is formin straight or branching
not branching; it's straight
86
what happens if formin is the only thing going on
just get long, straight actin filaments that form rapidly
87
what are long filaments from forming the building blocks for
loosely bundled filaments, tightly bundled filaments
88
what do formins build
actin stress fibers
89
what do formins nucleate
long actin filaments that are bundled into stress fibers
90
what do formins make
precursor straight filaments that go into long stress fibers
91
where are these long stress fibers
run underneath cell, responsible for generating majority of contractile forces
92
how do formin and profilin work together
form surfs growing plus end, elongating filament; gets hand off of actin monomers from activated profilin
93
what is anchored at minus end, arp2/3 or formin
ARP (not formin)
94
what does arp 2/3 form
branched filaments
95
what does formin form
straight filaments (surfs plus end as new monomers are added)
96
what else can profilin help besides formin
also feeds actin monomres to arp2/3
97
what is common denominator in creation of actin filaments
profilin
98
what is a way to regulate whether it's gonna grow or shrink
profilin
99
what is cofilin
disassembles old actin structure, gets monomers recycled back into cytoplasm to build new actin structures
100
where does cofilin bind
binds along actin filament like a copolymer
101
what does cofilin induce
binds, induces conformational change that puts filament under mechanical strain
102
what does putting filament under mechanical strain do
primes it to fall apart
103
basically what does cofilin do to shape
binds, gives it a twist, breaks it into pieces
104
describe appearance of filament when cofilin is there
thicker (cuz there's another protein there)
105
what does cofilin do to twists in the helix
shortens the twist in the helix, cuz its getting ready to fall apart into component pieces
106
what is lamellipodia generated by
arp 2/3
107
what are filopodia and stress fibers generated by
formin
108
what is cell cortex (forms mix of branched and unbranched filaments underneath plasma membrane) generated by
filamin
109
once filaments (long & straight) are generated by formin, what happens
fimbrin binds actin filaments into tight bundle
110
what does this tight bundle of actin filaments prevent
prevents addition of other proteins cuz there's not enough space
111
what is actin filaments and fimbrin
parallel bundle; tight packing prevents myosin II fron entering bundle
112
what type of structural actin bundle do we see in filopodia
parallel bundle; hella tight
113
what does alpha actinin do
longer cross linker that binds them with more space b/w adjacent filaments --> loose packing
114
what does the loose packing of a-actinin allow
allows myosin 2 to enter bundle
115
why is loose packing important
we wanna build an actomyosin stress fiber, so we need space for myosin motor proteins to enter network, bind actin, and act on them
116
describe actin filaments in filopodia/fimbrin mediated bundles
parallel
117
what does parallel mean for plus and minus ends
plus and minus ends are in same orientation
118
describe actin in alpha actinin
plus and minus ends alternate
119
what does filamin dimer do
cross links proteins but doesn't bundle them
120
what dooes filamin dimer do
hold them in a loose, flexible network
121
describe myosin II
dimer of 2 heavy chains, 4 light chains
122
what are heavy chains
green structures w/ globular head domain; this part of protein binds to actin
123
what do heavy chains have
long, flexible tails
124
what do the long tails form
coiled-coil alpha helix
125
how many heavy chains in every myosin II protein complex
2
126
how many light chains,
2 diff copies --> 4
127
where are light chains found
around neck/hinge region
128
what happens when myosin II is active
globular head domain can change conformation forward & backward as it walks along filament
129
what does neck/hinge region allow
flexibility
130
what is the 'business' part of protein
heavy chains w/ actin binding region
131
why are heavy chains the business region
it binds actin and converts chemical E into force
132
what are light chains
regulatory chains
133
what happens to light chains
they get phosphorylated or de-phosphorylated to tell myosin 2 to be active or inactive
134
what do heavy chains of the dimer do
mediate formation of bipolar filaments (many copies of bundled myosin II dimers)
135
what does regulatory light chain control
whether myosin 2 is just floating around cytoplasm as a dimer or polymerizing into larger assembly of bipolar filaments
136
what are bipolar filaments
many copies of myosin II heavy chains coming together in a specific orientation
137
what does phosphorylation of regulatory light chains control
whether they're single or form a filament
138
what do bipolar filaments have
mirror symmetry --> face each other (Helps generate force)
139
how do bipolar filaments lead to contraction & shorten network
motors walk on opposite directions on filaments; single bipolar filaments move actin filaments in opposite directions
140
what would happen if motors were all in same direction
filament would just slide left or right; filament would be moving not shortened
141
describe inactive state of myosin II
regulatory light chains aren't phosphorylated, myosin motor doesn't bind actin, coiled coil is held in conformation
142
basically what is inactive state
can't bind actin or other copies of myosin 2 motor
143
what are two upstream kinase that become activated
MLCK and ROCK
144
how are kinases activated
rhoA activates ROCK/MLCK, phosphorylates reg. light chains to activate myosin 2 activity & myosin bipolar filament assembly
145
what is the mechanism for contractility
something activates rhoA, rhoA activates ROCK/MLCK, triggers ^^ process leading to actomyosin contractility
146
what to both MLCK and ROCK have in common
both phosphorylate light chains leading to actin binding site being available; myosin tail released & can assemble w/ other copies to form bipolar filaments
147
why is myosin tail being released important
allows it to assemble other copies and form bipolar filaments
148
how does myosin 2 generate force
couples ATP hydrolysis to conformational changes
149
what does myosin-ATP cycle do
generates contractility important in generating forces and processes like cell migration & tissue shit
150
where does cycle start
myosin in its nucleotide free form --> attached to actin filaments
151
what comes next
ATP binds onto myosin head, induces release from actin
152
what happens to ATP
hydrolyzed, triggers conformational change that advances myosin head one monomer forward
153
now what is ATP in position to bind
the next actin monomer in the filament
154
what's the next step
inorganic phosphate is released (weak binding of head domain to new monomer)
155
what does weak binding trigger
ADP release, and power stroke
156
what is power stroke
where conformation goes backward, resetting to original position
157
basically sum up how myosin 2 works
starts bound to filament, releases filament, moves forward, binds filament, as its bound to filament it undergoes powerstroke to pull filament one monomer to the left
158
how many copies of myosin motor domain does bipolar filament have
100 copies
159
what generates contractile forces in cell
100 copies of myosin motor domain in bipolar filament all simultaneously doing this in opposite directions
160
what happens once ADP is released
back to nucleotide free state that is firmly anchored to filament waiting for next ATP to join
161
what does contractility apply to
stress fibers, cell migration, nuclear movement --> anytime force generated in a cell
162
what make actin interesting (structurally & functionally)
actin binding proteins
163
what does arp2/3 form
branched filaments (lamellipodia)
164
what does formin generate
single actin filaments that can be bundled
165
what does myosin II bipolar filaments contract
contract stress fibers
166
what activates myosin II
phosphorylation of myosin light chain
