BLOCK 3 Flashcards
1
Q
cytoskeleton
A
group of several different types of filamentous protein polymers
can form stable structures but is dynamic
2
Q
accessory proteins
A
control the assembly and function of cytoskeleton
3
Q
cytoskeleton properties
A
polar (not symmetric)
some structures are long lived, others are transient
can resist external forces and generate pushing or contractile ones
polymers can grow and shrink as subunits are assembled or disassembled
can rapidly reorganize in response to environment
4
Q
functions of cytoskeleton
A
cell morphogenesis
cell organization
cell division
cell adhesion
cell motility
5
Q
4 polymers in cytoskeletons
A
actin filaments (F-actin)
microtubules
intermediate filaments
septins
6
Q
what drives cell dynamics?
A
actin and microtubule cytoskeletons
7
Q
actin
A
most abundant protein in eukaryotic cells
1-20% of total proteins in cells
sequence has been conserved through evolution (actin is 80% identical in human and ameoba)
8
Q
actin in simple eukaryotes
A
one gene
9
Q
actin in mammals
A
several actin genes that produce multiple types of actin
a-actin in muscle
b-actin and y-actin in non muscle cells
10
Q
actin filaments
A
G-actin monomers bind ATP and compose F-actin helical polymers
11
Q
G-actin monomer
A
separated into two lobes by a cleft that binds ATP or ADP and Mg2+
12
Q
F-actin
A
helical polymer of G-actin subunits held together by non-covalent interactions
all oriented in the same direction – polar
13
Q
Barbed end of actin
A
+ end; elongates up to 10x faster than the pointed end
14
Q
pointed end of actin
A
- end; elongates slower than barbed end
15
Q
polymerization of actin in vitro
A
salts and G-actin
reversible
different than in the cell
16
Q
ways of monitoring actin polymerization
A
measuring the scattering of light (F-actin scatters more than G-actin)
pyrene-actin and spectrophotometry
visualizing filaments by fluorescence and EM
conducting sedimentation analysis (F-actin sediments more rapidly because larger than G-actin)
17
Q
pyrene-actin and spectrophotometry
A
attach fluorescent tag (pyrene) to actin which fluoresces more brightly when incorporated into AF-actin than in G-actin
18
Q
first step in actin polymerization
A
nucleation
19
Q
nucleation of actin
A
formation of a stable seed “nucleus” of three actin monomers which can elongate to form a filament
lag phase because is slow since actin dimers are unstable
20
Q
how to eliminate lag phase
A
add nucleating factors or actin filaments
21
Q
second step in polymerization
A
elongation
22
Q
elongation of actin
A
subunits add onto nuclei leading to growth. fast phase
23
Q
third step in actin polymerization
A
steady state
24
Q
steady state in actin
A
no net increase or decrease in amount of polymerized actin. elongation is balanced by shrinkage
25
critical concentration in actin
G-actin concentration in equilibrium with F-actin concentration
concentration of G actin at steady state
26
CC in actin in vitro
0.2uM
27
if the free subunit concentration is above CC
subunits will add onto the ends of filaments
28
if the free subunit concentration is below CC
subunits will be lost from the ends of filaments
29
how to demonstrate kinetics of actin filament ends
mix G-actin and F-actin with myosin S1 fragment to mark filament polarity. Newly assembled filaments are much longer at the plus end
30
ATP hydrolysis & CC
each actin monomer has an ATP that is hydrolyzed to ADP after its assembly into the polymer
ATP hydrolysis causes a conformational change that destabilizes the interactions within the filament
in the presence of ATP the two ends of an actin filament have different critical concentrations
31
CC+
0.12uM
32
CC-
0.6uM
33
at free actin concentrations >0.6uM
filaments grow at both ends
34
free actin concentrations <0.12uM
actin will shrink at both ends
35
free actin concentrations between 0.12uM and 0.6uM
filaments grow at + end and shrink at - end
36
actin treadmilling
**in the presence of ATP, actin will polymerize until the monomer concentration falls between the CCs of the two ends and will be treadmilling at steady state**
leads to flux of actin subunits through the filament
not in equilibrium because requires ATP hydrolysis
37
total actin concentration in cells
200uM
38
G-actin in cells
80uM
39
F-actin in cells
120uM
40
actin binding proteins (ABPs)
regulates assembly and disassembly of actin
41
actin sequestering proteins
maintains actin in monomer form by binding to monomers and preventing them from polymerizing
42
Thymosin B4
primary monomer sequestering protein
43
profilin
monomer binding protein that promotes G-actin to exchange ADP for ATP
binds + end of G-actin to inhibit initial nucleation but promotes actin polymerization onto existing filaments
44
thymosin B4 + profilin
compete to control growth of actin filaments
45
cofilin
interacts with and severs ADP-actin filaments leading to enhanced - end depolymerization of growth from new + ends
46
actin nucleating proteins (nucleators)
accelerate the initial kinetics of polymerization
and control cell shape, movement, and division during health and disease
47
formin
nucleators that quickly generate long unbranched filaments
dimerizes and facilitates barbed (+) end growth while remaining attached to the (+) end
humans have 15
48
Tandem actin monomer binding proteins
nucleate unbranched filaments
remain at (-) end during filament assembly
>7 in humans
49
ARP 2/3 complex
only actin nucleator that generates branched filaments
works with WASP family proteins to activate nucleation activity
causes formation of new actin filaments that is capped at its pointed (-) end by ARP2/3 but is free to elongate at barbed (+) end
50
phalloidin
prevents filament depolymerization (further polymerization is not affected)
51
cytochalasin
binds to + end of filaments and caps them; prevents elongation
CC shifts to - end and filaments eventually depolymerize
52
cytochalasin effects
blocks locomotion and cytokinesis; reversible
53
latrunculin
binds to actin monomers and prevents polymerization by sequestering them
causes rapid disassembly of actin filaments
reversible
54
microtubules
polymer of tubulin subunits in a cylindrical filament 24nm in diameter
55
subunit of microtubules
tubulin heterodimers
56
tubulin heterodimers
a-tubulin and b-tubulin
ab dimers do not come apart under physiological conditions
mammals have many a and b tubulin genes
57
protofilaments
has plus end and minus end
composed of stacked tubulin heterodimers
13 interact laterally to create a barrel for a microtubule
intrinsic polarity
58
cryo-EM
proteins frozen in liquid nitrogen vapor instead of the conventional fixation and heavy metal staining
59
ab tubulin heterodimers bind
GTP/GDP
60
B-tubulin ___ hydrolyze GTP to GDP
can
can also exchange GTP for GDP
61
A-tubulin ___ hydrolyze GTP to GDP
cannot
irreversibly binds to GTP
62
minus end (slow growing) of microtubule
ringed with a-tubulin
63
plus end (fast growing) of microtubule
ringed with b-tubulin
64
MTOC / centrosome
inside the cell the minus ends of microtubules are usually capped and embedded here
65
microtubule dynamics in vitro
polymerization of pure tubulin initiated by raising temperature to 37 degrees and depolymerization at 4 degrees
66
tubulin polymerization monitoring
measuring scattering of light (polymer scatters more than monomer or dimer)
attach fluorescent tag (rhodamine or fluorescein) to tubulin
67
microtubule assembly kinetics
similar to actin
68
at tubulin concentrations below CC
polymerization does not take place
69
tubulin concentrations above CC
polymerization is induced and microtubules assemble until free tubulin concentration falls to CC and steady state is reached
70
elongation of MT
GTP bound ab-tubulin heterodimers add onto + ends of MTs
B-tubulin hydrolyzes GTP to GDP after it is incorporated into the MT
71
the growing (+) end of the microtubule will contain
GTP B-tubulin
72
the bulk of the microtubule will contain
GDP B-tubulin
73
GTP cap
GTP b-tubulin on + end of mirotubule
74
how can the GTP cap be lost?
dissociation of GTP subunits from the MT end or by GTP hydrolysis
loss of GTP cap destabilizes the polymer
75
why does loss of the GTP cap destabilize the polymer?
GTP tubulin more readily makes lateral interactions with other protofilaments that maintain cylindrical structure of the end
if the MTs have a GDP cap these connections are weakened and the protofilaments tend to splay apart
76
protofilament splaying
destabilizes the MT and leads to depolymerization or shrinkage
off rate of GDP tubulin is high and disassembly of the MT can be rapid and can result in complete disassembly of that MT
77
catastrophe
complete disassembly of MT after loss of GDP cap
78
rescue
MT regrowth after catastrophe
79
individual microtubules have dynamic instability
rapidly change length
80
dynamic instability
alternation between growing and shrinking states occur randomly and is due to the conversions between a GTP and a GDP cap at the plus end
at steady state, total amount of polymer in bulk solution of MT is constant but any individual MT can be either elongating or shortening
81
total tubulin concentration in cells
10-20uM
82
cc of tubulin in cells
0.03uM
83
why do microtubules not form randomly in cells if polymerization is highly favored from their Ccs?
kinetic barrier of nucleation
84
microtubule-associated proteins (MAPs)
aids microtubule assembly
85
microtubule organizing center (MTOC)
nucleates and organizes cellular microtubules
MTs radiate from here
86
centrosome
MTOC in interphase cells
consists of a pair of centrioles surrounded by a pericentriolar material
87
MTs in interphase cells
minus end of the MT is embedded in the centrosome and plus end faces the cytosol
88
taxol
binds MTs and stabilizes the polymer, preventing disassembly
blocks MT dependent processes (mitotic spindle assembly)
89
colchicine
binds to tubulin dimer so when it polymerizes, further polymerization is prevented on the MT end
promotes formation of a GDP gap and MT destabilization
reversible
90
nocodazole
binds to tubulin subunits and triggers depolymerization
reversible
91
pericentriolar material (PCM)
MT minus ends embedded here
92
y-TURC
protein complex of y-tubulin and other proteins; anchors MTs at the centrosome
nucleates assembly of pure tubulin
93
y-tubulin
nucleates microtubules at the centrosome
conserved in all eukaryotes and is distinct from ab-tubulin
does not polymerize itself but exists in a protein complex called y-TURC
94
ABPs
control assembly, disassembly, organization, and movement of actin filaments
95
formins
bind monomers and filaments
96
ARP2/3 complex
binds monomers and filaments
97
Thymosin b4
binds monomers
98
profilin
binds monomers
99
latrunculin
actin; depolymerizes by binding actin subunits
100
cytochalasin B
actin; depolymerizes by capping filament plus ends
101
phalloidin
actin; stabilizes by binding along filaments
102
taxol
MT; stabilizes by binding along filaments
103
nocodazole
MT; depolymerizes by binding tubulin subunits
104
colchicine
MT; depolymerizes by capping filament ends
105
Actin binding proteins (ABPs)
control assembly, disassembly, organization, movement of actin filaments
106
capping proteins
stabilize filaments by binding to their ends
| stabilized bc prevents addition or loss of subunits from ends
107
CapZ
capping protein; caps the + end of filaments
108
tropomodulin
caps - end of actin filaments
109
stabilizing proteins
bind along length of filaments
110
tropomyosin
binds subunits along filaments and stabilizes polymerized state
111
severing/depolymerizing proteins
disassemble actin filaments and higher order structures
112
cofilin
enhances rate of actin depoly by severing actin filaments and creating more (-) ends
113
gelsolin
severs actin filaments and breaks them into short fragments but remains bound to (+) end (activated by calcium)
114
cross-linking proteins
have two actin-binding domains separated by spacer domains to produce assemblies of different types
115
filamin and spectrin
cross linking proteins with longer, more flexible spacers
arrange filaments in gel-like networks
116
fimbrin, vilin, fascin
cross linking proteins with shorter, more rigid spacers
organize filaments into aligned bundles (and lead to tight bundles)
117
a-actinin
crosslinks filaments into bundles that are arranged into parallel and antiparallel arrays for contraction
118
membrane-actin linker proteins
attach actin cytoskeleton to the plasma membrane
interactions between integral membrane proteins and actin filaments
119
ERM proteins
act as linker between F-actin and integral membrane proteins
120
spectrin
connects actin to membrane proteins
121
dystrophin
links cortical actin to muscle cell membranes via dystroglycan complex
122
cell cortex
actin-rich layer beneath plasma membrane
gives mechanical strength and allows surface movments
123
spectrin-based membrane skeleton
lies immediately adjacent to plasma membrane
124
ezrin
links bundled actin to the membrane in microvilli
125
hereditary spherocytic anemias
mutations in genes encoding proteins of RBC membrane skeleton (spectrin, ankyrin, band 4.1)
rupture easily
126
muscular dystrophies
genetic; weakening of skeletal muscle
127
Duchenne muscular dystrophy (DMD)
dystrophin is messed up; plasma membrane of muscle cells weaken and eventually rupture
128
Microtubule associated proteins (MAPs)
control assembly, disassembly, organization, and movement of microtubules
129
tubulin oligomer binding proteins
stathmin can bind and sequester tubulin heterodimers or oligomers and promote catastrophe and depoly
130
nucleating proteins
y-TURC nucleates MT assembly at centrosome
131
end binding proteins
+TIPS bind to the (+) end of MTs
132
+TIPS
EB1
| CLIP-170
133
EB1
+TIP that regulates (+) end dynamics and can link MTs to organelles
134
CLIP-170
+TIP that mediates interaction of chromosomes and membranes with (+) end
135
severing proteins
severs MTs and promotes depoly at (-) ends
katanin
136
katanin
severing protein
137
depolymerizing proteins
a subset of kinesins promote MT depolymierzation at (+) end by binding to and inducing protofilament curling
138
kin13
depolymerizing protein
promotes depolymerization of GTP-tubulin and does not involve hydrolysis of GTP cap
139
polymer-binding MAPs
stabilize MTs by binding to their sides
enhance assembly by stabilizing nuclei
organize MTs into bundles
mediate Mt interactions with other proteins
140
polymer-binding MAPs domains
MT binding domain
projection domain
141
MT binding domain of polymer-binding MAPs domains
binds several tubulin dimers at once and helps stabilize polymer
142
projection domain of polymer-binding MAPs domains
interacts with MTs or other structures such as IF
143
Tau
organize MTs in neuronal axons and dendrites
144
MAP2
organize MTs in neuronal axons and dendrites; spacing bw MTs is greater than in Tau expressing cells
145
Tau aggregation in an insoluble form
involved in Alzheimer's disease pathogenesis
146
myosin motor proteins
move along actin filaments by coupling the energy from ATP hydrolysis to conformational changes
mechanochemical enzymes
147
most myosins are ____ end directed motors
(+)
148
myosin composition
heavy and light chains
149
heavy chain of myosin
head, neck, and tail domains
150
head (motor) domain of myosin
actin binding and ATPase activities
151
neck domain of myosin
regulatory function, site of attachment for regulatory and essential light chains or calmodulin
152
tail domain of myosin
differ depending on properties
153
myosin II
first described as motor protein in muscle that powers contraction
2 heavy and 2 light chains
tails mediate polymerization of myosin II into bipolar thick filaments
154
actin filament sliding assay
motor function of myosin is demonstrated by filament sliding assays
myosin molecules stuck to glass, add actin filaments
155
ATP hydrolysis is coupled to myosin ____
motility
156
myosin ATPase activity is _____
actin-activated
157
myosin-actin cross-bridge cycle: rigor state
ATP binding site is empty and myosin is tightly bound to actin
0
158
myosin-actin cross-bridge cycle: ATP binding
when ATP binds to myosin, ATP binding cleft closes, opens actin binding cleft and weakens interaction with actin
1
159
myosin-actin cross-bridge cycle: ATP hydrolysis
after detaching from actin filament ATP is hydrolyzed to ADP and pi
conformational change and causes head to move to new position before rebinding the filament
2-3
160
myosin-actin cross-bridge cycle: Pi release
pi released and myosin head changes
POWER STROKE
exerts force on actin filament causing it to move
restoring to rigor conformation
4
161
myosin-actin cross-bridge cycle: ADP release
after ADP is released, myosin remains in rigor state and ATP exchange releases the head from actin
5
162
myosin I
shorter tail domains, single-headed, do not assemble into filaments
163
myosin I tails
mediate movement of filaments past one another
attach to and move vesicles or organelles
164
Myosin V
dimers; vesicle transport
165
which myosins have conserved head domains with variable tail regions?
III, IV, VI-XV
166
myosin VI
moves towards (-) end of F-actin
167
myosin V dimrs
walk along F-actin
processive movement: one head always in contact with the actin
168
myosin VI mutation
deafness in mice
169
myosin VII mutations
deafness and blindness in humans
170
two classes of MT motor proteins
kinesins and dyneins
171
kinesins
MT-activated mechanochemical ATPases
172
3 classes of kinesins
Kin-N
Kin-C
Kin-I
differ in location of motor domain in primary sequence of protein
173
Kin N kinesins
motor domain at N-terminus
+ end directed
174
Kin C kinesins
C terminal motor domain
(-) end directed
175
Kin I kinesins
internal motor domain
do not move along MTs but bind MT ends and promote protofilament peeling
176
conventional kinesin
two heavy chains, two light chains
motor head at N term of heavy chain
neck domain
a-helical coiled-coil tail domain
kinesin light chains mediate interaction of kinesin with membrane vesicles
177
microtubule gliding assay
demonstrates motor function of kinesin
178
kinesin cross bridge cycle
similar to myosin
179
processive motility of kinesin motor proteins
movement over long distances without dissociating
important for vesicle and organelle transport
180
dyneins
enzymes that couple the energy from ATP hydrolysis to (-) end directed movement along MTs
2-3 heavy chains
181
two types of dyneins
cytoplasmic
| ciliary/flagellar
182
dynactin complex
links dynein to cargo
critical for cytoplasmic dynein
mediates attachment of dynein to vesicles and organelles
183
charcot-marie-tooth disease and kidney diseases
kinesin deficiencies
184
chronic infections of respiratory tract due to nonfunctioning cilia
dynein deficiencies
185
ALS
mutation in dynactin complex
186
where are the microtubule + ends
edge of cell
187
where are the - ends in MT
in centrosomes
188
are kinesins or dyneins important for melanosome localization in high cAMP?
kinesins
189
are kinesins or dyneins important for melanosome localization in low cAMP?
dyneins
190
what will melanosome organization look like if you treat cells with taxol?
MTs stabilized and grow at plus ends
191
actin functions
membrane protrusion
cell adhesion
endocytosis and trafficking
cytokinesis
192
actin functions: membrane protrusion
lamellipodia and filopodia
193
actin functions: cell adhesion
stress fibers and focal adhesions
194
actin functions: endocytosis and trafficking
clathrin pits and endosomes
195
microtubule functions
organelle positioning
anterograde transport
retrograde transport
mitosis
196
microtubule functions: organelle positioning
ER and golgi
197
microtubule functions: anterograde transport
ER to golgi to plasma membrane
198
microtubule functions: retrograde transport
Endosome to golgi to ER
199
lamellipodia and filopodia
membrane protrusions that move adhere to substrate
have stress fibers that function in cell adhesion and contraction
200
fibroblast
cell of connective tissue
201
lamellipodia
thin sheet like structures of fibroblast
202
filopodia
thin needle-like projections or spikes on fibroblast
203
what happens when you microinject cells with fluorescently labeled actin?
actin is incorporated into filaments at extreme leading edge and polymerization takes place there
204
what drives membrane protrusion?
assembly of actin filaments
205
retrograde flow
after actin filaments are nucleated, the network of filaments moves backward to interior of cell
cofilin is the major depolymerizing activity behind the leading edge
206
actin filament orientation
(+) ends facing membrane in the direction of protrusion
207
lamellipodia actin filament organization
branched
208
filopodia actin filament organization
parallel bundles
209
stress fibers
long bundles of actin filaments that lie along the lower surface of the cell
ends terminate at focal adhesions
210
focal adhesions
transmembrane structures that attach cell to underlying substrates
211
how are dynamic actin specializations triggered to form?
small G protein class: Rho GTPases
212
Rho-family GTPases
Rho
Rac
CDC42
213
Guanine nucleotide dissociation inhibitors (GDIs)
stabilize inactive GDP-bound form
214
WASP family proteins interacting with Rho-family GTPases
WASP proteins are actiavted by small GTPases to assemble actin in membrane protrusions
usually is autoinhibited but Cdc42-GTP binding relieves the autoinhibition
crucial for signaling cascades to activate WASP / formins
215
microinjection of Rac-GTP
formation of lamellipodia and membrane ruffles
216
microinjection of Cdc42-GTP
formation of filopodia
217
microinjection of Rho-GTP
formation of stress fibers
218
what does actin cytoskeleton direct?
endocytosis endosome trafficking, endosome rocketing, and membrane sorting
219
what does branched actin assembly facilitate?
endocytic internalization
220
what does MT cytoskeleton direct?
organelle positioning, membrane transport, secretory pathway
221
how does golgi positioning depend on MT?
golgi is usually near the centrosome
MT position golgi near ER
222
stress fibers contract in response to phosphorylation of non-muscle myosin II light chain by MLCK
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