lecture 9 Flashcards
1
Q
besides actin what are 3 other cytoskeletal systems in cells
A
microtubules, intermediate filaments, septins
2
Q
what is associated w/ lamellipodia
A
Arp 2/3
3
Q
major function of intermediate filaments
A
provides structure and strength to cells and tissues
4
Q
what can intermediate filaments connect to
A
actin
5
Q
are microtubules dynamic
A
hella dynamic
6
Q
where are minus ends of microtubules
A
found at centrosomes, where they are nucleated
7
Q
where are plus ends of microtubules
A
radiate away from centrosomes, towards leading edge of this migrating cell (in this example)
8
Q
what do plus ends of microtubules do
A
program where the leading edge is gonna go
9
Q
what would happen if microtubules were growing in a diff direction
A
leading edge would switch and re-position to where microtubules were found
10
Q
what is polarity of microtubules important for
A
dictating overall polarity of cell
11
Q
describe polarity of microtubules
A
minus ends toward cell center, plus ends toward leading edge of cell plasma membrane
12
Q
why do microtubules have dramatic effect on cell structure & function
A
plus ends direct where signaling molecules go & are activated –> generates lamellipodial actin that makes leading edge AND tracks for vesicles w cargo (toward leading edge)
13
Q
what 2 things does polarized microtubules lead to
A
polarized signaling and polarized trafficking/secretion (both important for cells to move in a certain direction)
14
Q
what is the brains of operation
A
microtubules
15
Q
are intermediate filaments rlly dynamic
A
not really; kinda slower turnover
16
Q
what do intermediate filaments do
A
provide structural strength & form physical barriers
17
Q
what important thing can intermediate filaments do
A
can segregate one part of cytoplasm from another, form a cytoskeletal wall (thru bulk)
18
Q
are intermediate filaments static or dynamic
A
relatively static (compared to microtubules & actin)
19
Q
are intermediate filaments polarized
A
no; chemically identical
20
Q
can intermediate filaments be used to traffic things?
A
no, because there’s no inherent directionality, nothing to tell motors moving vesicles which way to go
21
Q
what else do intermediate filaments do
A
provide structural strength
22
Q
what are 3 chemicals that affect microtubules
A
taxol, nocodazole, colcichine
23
Q
what does taxol do
A
stabilizes microtubules, locking them into that configuration
24
Q
what is a medical application of taxol
A
chemotherapy; kills rapidly dividing cancer cells by stabilizing & disrupting their microtubules
25
what do nocodazole and colchicine do
cause microtubule depolymerization --> cause them to fall apart
26
which chemicals depolymerize
nocodazole, colchicine
27
which chemicals stabilize
taxol
28
which is worse, stabilizer or depolymerizer of microtubules
both are equally bad; b/c dynamics (growing AND shrinking) are essential for health and function
29
what is microtubule subunit
tubulin protein
30
how does actin float around as
monomer
31
how does tubulin exist
as a heterodimer
32
how many genes is tubulin expressed from
3 diff genes --> 3 diff proteins
33
what are 2 distinct proteins in tubulin
alpha tubulin and beta tubulin
34
what are building blocks that create microtubules built up from
stable heterodimers that consist of 1 b-tubulin bound to 1 a-tubulin
35
are you ever gonna find alpha and beta tubulin by themselves?
no; always together in cytoplasm
36
what polymerizes to form the large diameter microtubules
tubulin heterodimer
37
describe association/interactions of actin that give rise to helical filaments
head to tail associations
38
describe associations of alpha-beta tubulin
head to tail interactions AND lateral associations w/ neighboring heterodimers
39
what do these lateral associations allow microtubules to do
not just make filament structure but also the hollow tube that is fully formed microtubule
40
what gives rise to hollow cylinder that is microtubule
head to tail associations AND lateral associations
41
what does plus end bind to
minus end
42
how do alpha and beta tubulin bind to
plus end of beta tubulin, minus end of beta tubulin binds to plus end of alpha tubulin, minus end of alpha tubulin (at bottom of screen)
43
what nucleotides are tubulin heterodimers bound to (like actin monomers)
guanine
44
where are nucleotides located on tubulin heterodimer
one copy at plus end of beta tubulin, second copy at plus end of alpha tubulin
45
what happens to nucleotide (GTP) on beta tubulin
hydrolyzes after joining the growing filament
46
what happens to nucleotide on alpha tubulin
buried in the dimer; has no role in subsequent filament dynamics
47
when we talk about GTP hydrolysis, what does it refer to
nucleotide on beta tubulin
48
is this (GTP to GDP) a hydrolysis or exchange
hydrolysis, not an exchange
49
what has activity to hydrolyze GTP to GDP, and how is it triggered
tubulin has the activity, and it's triggered by polymerization (like actin)
50
what happens after tubulin is added to a filament
internal chemical timer starts, after milliseconds it is hydrolyzed to GTP
51
what does GTP form like
it likes being in a filament, favors polymerization
52
what does GDP form like
doesn't wanna be in filament anymore (like actin)
53
what does the ATP to ADP switch in actin do
gives rise to treadmilling
54
what does hydrolysis of GTP to GDP in microtubules do
dictates whether it's gonna be growing or switching to shrinking (dynamic instability)
55
what is dynamic instability, and what is it a direct result of
ability for MT to switch from growing to shrinking to growing again; result of hydrolysis of GTP on beta tubulin
56
do we need to worry about minus end of microtubules
no; everything is plus end, can ignore minus end b/c minus ends are all anchored into centrosomes where they are nucleated
57
in a rapidly growing microtubule what is there at plus end
GTP cap
58
when does GTP cap form
growing polymer, where heterodimers are added so fast they don't have time to hydrolyze GTP to GDP (unlike the other side who has been there longer)
59
what is length of filament correlated w/
how long heterodimers have been in filament
60
what does it mean as long as there's abundant GTP bound heterodimers to be added to plus ends
it's gonna keep growing
61
how does it lose GTP cap
supply of GTP bound heterodimers runs low (whether random or regulated), not added as quickly, they have time to hydrolyze GTP to GDP before another heterodimer is added --> loss of GTP cap
62
what happens after loss of GTP cap
all becomes GDP tubulin, which likes to fall apart (doesn't like to be polymerized in a MT --> rapid collapse)
63
what does GTP cap do
holds the microtubule together, allows more heterodimers to be added
64
describe dynamic instability in context of GTP/GDP
loss of GTP cap means you can't add GTP bound monomers as quickly, so filament switches from growing to rapidly shrinking
65
is dynamic instability reversible?
no; doesn't lead to destruction of entire molecule
66
how can it switch b/w growing and shrinking
linked to [ ] of GTP bound heterodimers; if more GTP bound MTs are available, goes from shrinking to growing
67
how do MTs switch from growing to shrinking and back
based on availability of GTP cap
68
what is catastrophe
switches from growing to shrinking
69
what is rescue
switches back to growing [enough GTP bound heterodimers added --> GTP cap is established]
70
what is GTP bound to
beta tubulin in polymerized form
71
what happens when the heterodimer is released from filament
it takes GDP with it
72
why is rescue, catastrophe, growing/shrinking so important
MTs need to get to different spots quickly, need to sample diff areas/parts of cell {being stuck in one spot is bad}
73
what does growing shrinking allow
can go from where leading edge is to where leading edge is gonna be next --> key to their ability to steer & control cells, organization, cell function
74
why does GDP form of tubulin wanna leave polymer so badly
after hydrolysis, subtle shift in conformation that weakens bond b/w adjacent heterodimers (can see slight curvature to filament)
75
what does conformational change do
decreases affinity of adjacent heterodimers for each other (curve)
76
what happens once GTP cap is lost
they are primed to fall apart
77
what happens once heterodimer is released
GDP to GTP exchange in cytoplasm, recycled and ready to be reincorporated
78
what does the GTP cap represent
high affinity interactions; want to stay together, rapidly growing MT
79
what does losing cap represent
weakened affinity, due to chemical shift (change from GDP to GTP), just fall apart
80
how does it fall apart?
chunks peel away as it shrinks, not individual monomers)
81
what is crucial for MT function
ability to switch from growing to shrinking and back again
82
what dictates whether you're growing or shrinking at plus end
rate of addition of monomers
83
what end of MT is this going on
all at plus end
84
how many monomers does actin oligomer need
3
85
how many tubulin heterodimers needed
7!!!! also needa come together in a spiral structure
86
what machinery helps overcome these challenges/conditions
template that holds tubulin in exact right position --> can have heterodimers at right place & time, so you can add new heterodimers to get that growth
87
why do you need a protein cmplex
need it to nucleate new filaments by holding blocks in scaffold, so you don't need to take time for them to randomly come together in right orientation
88
what is the 3d tubulin
gamma-tubulin small complex
89
what are first 2 tubulins
alpha and beta tubulin which forms heterodimer --> gonna get added to growing fliament
90
where is gamma tubulin found
only at sites of microtubule nucleation
91
what does gamma tubulin form
scaffold that additional alpha-beta tubulins can be added to
92
how many actin homologs in arp 2/3
2 actin homologs
93
how many tubulins for gamma tubulin
7 copies --> 14 tubulins held in right place to initiate spiral structure
94
what is job of gamma tubulin small complex
initiate spiral structure that alpha and beta tubulin can be added to, to get a functioning microtubule
95
what is the only place that gamma tubulin has a function in cell
here; sites of MT nucleation
96
what do accessory proteins do
hold them at right location, makes sure it happens at centrosome
97
where specifically are gamma tubulin small complexes found
centrosomes
98
where are all MTs growing out from
one locatino
99
what is centrosome packed w/
packed w/ gamma tubulin ring complexes
100
what is centrosome
pair of centrioles, duplicated during cell division & evenly distributed among daughter cells
101
what is centrosome / pair of centrioles surrounded by
pericentriolar material
102
what do gamma tubulin small complexes bind to
pericentriolar material
103
where do MTs grow away from
centrosome
104
where are plus and minus ends
plus ends grow away, minus ends are attached & locked to centrosome
105
where is minus end gonna be always
anchored to centrosome via gamma tubulin nucleating complex (unless it depolymerizes totally)
106
what 3 proteins control microtubule dynamics (contribute to whether it grows or shrinks)
stathmin, kinesin-13, XMAP215
107
what is stathmin
analogous to thymosin for actin; grabs & binds heterodimers, prevents them from being added to plus end
108
what dose stathmin promote
shrinking; catastrophe
109
what is kinesin-13
microtubule motor; don't walk along MTs, but use thats systems to rip them apart & induce catastrophe
110
what are kinesins
protein complexes that use power of ATP hydrolysis to walk along MTs
111
how many copies of Kinesin-13 do we see on plus end of MT
4 copies on plus end of MT
112
what is XMAP215
plus end protein that does opposite --> stabilizes plus ends, harder time falling apart
113
when does XMAP215 stabilize plus end until
GTP tubulin recovers and keeps on going
114
what is plectin
protein that connects microtubules (also actin) to other structures -> intermediate filaments
115
what does plectin do specifically
connects MTs to intermediate filaments
116
what do catastrophe & stabilization factors do
both target GTP-bound tubulin dimers at plus end of polymer
117
what does kinesin 13 do
induces depolymerization (even if cap is there)
118
what happens if cell needs MT to shrink but doesn't have time to get rid of GTP cap
kinesin 13!!!
119
what is XMAP215
opposite; accelerates growth & promotes MT polymerization
120
what does stathmin do
binds to heterodimers & prevents them from being added to plus end
121
what happens if you have a bunch of active stathmin around
reduces [ ] of available heterodimers, you get catastrophe & shrinking
122
what does thymosin bind to
1 actin monomer
123
what does stathmin bind to
2 heterodimers
124
what motor protein for actin
myosin 2 (generates contractile force)
125
what motor protein for MTs
kinesin 1
126
what do motor proteins do for MT
move cargo from one end of cell to another
127
describe kinesin 1
looks like myosin 2 (long tail that wraps around neighbor --> extended coil coil)
128
what is structure of kinesin
functional dimer, 2 globular motor domains
129
what are globular motor domains responsible for
responsible for binding & unbinding to microtubule filament to walk from minus end toward plus end
130
what does kinesin 1 mean for vesicles
you have a vesicle, starts at center of cell, moves toward cell edge
131
how can you infer kinesin 1 is dragging it along a polarized MT or centrosome
go from where minus ends are to cell membrane where plus ends are
132
what is mechanism for kinesin 1
driven by ATP hydrolysis
133
where is leading head closer to
closer to plus end (b/c that's drxn MT is walking)
134
what is next step
leading head is bound ADP, lagging head is bound to ATP
135
what happens after ATP is hydrolyzed on lagging head
phosphate is released from lagging head --> lagging head is released, conformational change at neck region of motor protein, leads to lagging head stepping over leading head and essentially becomes leading head
136
what switches
leading head becomes lagging head
137
what happens at the end
end up in exact same spot where you started cycle; leading head is ADP bound, lagging head ATP bound
138
what does hinge region have
flexibility, allows them to step over each other
139
describe myosin 2
contracts filaments in opposite drxns b/c heads are facing each other in bipolar filaments
140
what is stationary and what is moving in myosin
myosin motor protein is stationary, actin is slid/moving
141
what is stationary and moving in MT
microtubule is stationary, motor protein is walking on it
142
which 2 motor proteins move along MTs
kinesin 1, (cytoplasmic) dynein
143
is dynein smaller or bigger than kinesin
way bigger & complicated
144
what is kinesin
motor domains that walk along filament, long tails are connected to vesicles/cargo
145
what are kinesins doing/have as they walk towards plus end filament
carrying something w/ them
146
what about dynein
binds to cargo, carries cargo on one end, other end walks along microtubule
147
describe dynein
cargo bound motor protein, goes from plus end to minus end
148
what if cell wants to get stuff into cell (endocytic event)
gonna attach to dynein so it can walk opposite drxn where all minus ends are in cell's nucleus
149
basically what to know about dynein
cargo carrying motor protein that goes to minus ends of MTs
150
where does kinesin walk to
walks toward plus end of MTs
151
first step of intermediate filament assembly
start w/ intermediate filament monomer, forms alpha helix --> has amino terminus (N terminus) and carboxy terminus (C terminus) like any other protein
152
what is next step
2 monomers come together to form a coiled coil dimer (alpha helices wrap around each other to form coiled-coil)
153
what happens when coiled coil dimer is formed
amino termini are lined up, carboxy termini are lined up --> dimers are also polarized
154
next step
2 dimers come together to form a staggered tetramer --> polarity is lost
155
described the tetramer
non-polarized
156
what happens next
lateral association of 8 tetramers. (unpolarized)
157
what happens once 8 tetramers come together
can be added to growing filament
158
what is final result at end of the day
non-polarized filament, no plus or minus end
159
why would a motor protein not know where to go
b/c no directionality; that's why there's no known motor proteins that interact w/ intermediate filaments
160
why are intermediate filaments so strong and hard to break
woven together like a rope
161
describe associations of intermediate filaments
head to tail associations but many MORE lateral associations --> can stretch w/o breaking
162
describe associations of actin filaments
head to tail monomer formation
163
describe associations of MTs
head to tail w/ some lateral associations
164
describe defect/mutation in intermediate filaments (keratin)
lost structural integrity; if they rub hands together, form blisters b/c no resistance to force
165
describe dynamic of intermediate filaments
dynamic but slower than actin
166
what does plectin do
links MTs and intermediate filaments --> hybrid cytoskeletal structure
167
what is one function of cytoskeletal cross-linking
connects diff parts of cells into one continuous mechanical network
168
what is intermediate filaments in nuclear lamina
line inner surface of nuclear envelope, gives nucleus structural strength
169
what are cross linking proteins that connect nuclear skeleton to cytoskeleton in cytoplasm
KASH and SUN proteins
170
what to know
when you look at cell, there's interconnected filament system from nuclear skeleton to cytoskeleton bridged by KASH and SUN domain containing proteins
171
on test, ex. of why you'd want cytoskeletal elements to be connected
top is fibroblast that's migrated thru dense ECM (filamentous protein network that's glue that holds tissues together)
172
what do fibroblasts have to do
needa navigate dense env. to get to wounds and heal them
173
what is biggest issue when cell has to move thru dense env.
getting bulky nucleus thru tight spaces (don't want it to rupture)
174
in lab what is vimentin IF wrapped around
wraps around nucleus
175
what is actomyosin machinery that generates majority of cellular forces is [ ] in
[ ] in cytoplasm, connects to IFs
176
what is actomyosin contractility physically transmitted to
transmitted to IFs via plectin cross-links
177
how do you get force from actomyosin contractility to intermediate filaments (vimentin IFs is the tow cable)
b/c they're bound to plectin
178
what do actomyosin and IFs together allow
solve problem of getting bulky nucleus thru tight spaces
179
what do septins form
filaments, highly enriched under nucleus [influence microtubule & actin function, form filament, sheets, rings]
180
what can septins form
diffusion barriers that divide cell
181
what do plectins do
connect IFs to other cytoskeletal structures
