Cytoskeleton Flashcards
What are the 3 components of the cytoskeleton?
microtubules
microfilaments
intermediate filaments
Briefly describe the structure and components of microtubules
structure: hollow, thick, rigid, unbranched
components: tubulin polymers (dimers of alpha and beta tubulin)
Briefly describe the structure and components of microfilaments
structure: solid, thin, flexible, branched, helical
components: actin polymers
Briefly describe the structure and components of intermediate filaments
structure: tough, rope-like
components: 70+ different proteins
What cell types are microtubules in?
all eukaryotes
What cell types are microfilaments in?
all eukaryotes
What cell types are intermediate filaments in?
animals only
Where are microtubules located?
cytoplasm
Where are microfilaments located?
cytoplasm
Where are intermediate filaments located?
cytoplasm and nucleus
Compare the diameter of microtubules, microfilaments, and intermediate filaments
microtubules: 25 nm
microfilaments: 8 nm
intermediate filaments: 10-12 nm
Which of the 3 cytoskeleton is the thinnest? which is the thickest?
thinnest = microfilaments
thickest = microtubules
What monomers make up microtubules?
alpha tubulin
beta tubulin
What monomers make up microfilaments?
actin
What monomers make up intermediate filaments?
varies
What is the enzyme activity in microtubules?
GTPase
What is the enzyme activity in microfilaments?
ATPase
What is the enzyme activity in intermediate filaments?
trick question! there’s no enzyme activity
Is there structural polarity in microtubules?
yes
Is there structural polarity in microfilaments?
yes
Is there structural polarity in intermediate filaments?
no
What motor proteins function in microtubules?
kinesins and dyneins
What motor proteins function in microfilaments?
myosin
What motor proteins function in intermediate filaments?
none!
Where does growth occur on microtubules?
+ end
Where does growth occur on microfilaments?
+ end
Where does growth occur on intermediate filaments?
internal
What are 5 functions of the cytoskeleton?
strength and structural support
internal organization of cellular components
allows cells to interact with each other and the environment
allows some cells to change shape and move
allows cells to rearrange their internal components as they grow, divide, and respond to external signals
What are the main functions of microtubules?
structural support
intracellular transport
spatial organization of organelles
cell motility
cell division
What are the main functions of microfilaments?
intracellular transport
cell motility
cell contractility
cell division
What are the main functions of intermediate filaments?
structural support
mechanical strength
What 5 general similarities do microtubules and microfilaments have?
polarity
nucleation
elongation
NTP hydrolysis
+ end caps
Briefly describe why microtubules and microfilaments have polarity in common
they both have + and - ends
growth is more common on the + end
Briefly describe why microtubules and microfilaments have nucleation in common
Both have a ‘lag’ phase - a slow process to form the initial aggregate
Briefly describe why microtubules and microfilaments have elongation in common
they both see rapid growth after the aggregate forms
Briefly describe why microtubules and microfilaments have NTP hydrolysis in common
Both hydrolyze an NTP (ATP for actin, GTP for tubulin) into NDP + Pi to incorporate incoming units at the + end
Briefly describe why microtubules and microfilaments have + end caps in common
Both have + caps that indicate elongation (they are bound to NTP and added at the + end)
Describe the polymer polarity. Which cytoskeleton elements have this?
unique + and - ends on microtubules and microfilaments
What occurs at the + end of microtubules and microfilaments?
addition (growth) of new units (tubulin dimers on microtubules and actin monomers on microfilaments)
What is added to the + end of microtubules?
tubulin dimers
What is added to the + end of microfilaments?
actin monomers
describe nucleation
the formation of an initial aggregate of individual units (tubulin dimers or actin monomers)
Is nucleation slow or fast initially? why?
slow because the first few units are not very stable (they lack lots of subunit-subunit interactions)
What happens once the initial aggregate has formed?
elongation (rapid growth) is much faster
What does polymerization eventually reach over time? when does it reach this?
an equilibrium phase or steady state when the growth of the polymer is equal to the shrinkage of the polymer
What does actin bind to?
ATP
What does tubulin bind to?
GTP
What happens to new units that join the + end of a polymer?
they bind to either ATP (actin) or GTP (tubulin)
What happens to the NTP that has bound the new unit at the + end of a polymer?
it is hydrolyzed
T or F: the older units of a polymer are bound to ATP/GTP
false! the NTP they are bound to is hydrolyzed to ADP/GDP
Describe the + end caps
when a polymer is extending, a ‘cap’ of newly added ATP/GTP-bound units are at the plus end
What does the presence of a + cap signify?
elongation of the polymer
What are microtubules composed of?
heterodimers of alpha and beta tubulin
In microtubules, what is alpha tubulin bound to?
GTP
T or F: the GTP that alpha tubulin binds to is hydrolyzed
FALSE it is never hydrolyzed
In microtubules, what is beta tubulin bound to?
either GDP or GTP
On a microtubule, where will alpha tubulin be located? What does this contribute to?
On the bottom or beneath a beta tubulin
contributes to structural polarity
On a microtubule, where will beta tubulin be located? What does this contribute to?
on the top or above alpha tubulin
contributes to structural polarity
How are alpha-beta tubulin heterodimers oriented in microtubules? What does this form?
head-to-tail in 13 staggered protofilaments to form a hollow tube
Describe protofilaments
the way alpha-beta tubulins are organized to form the hollow microtubule
How many protofilaments are required to make a microtubule?
13
Are microtubules hollow or solid?
hollow
In a microtubule, what forms the plus end?
exposed Beta tubulins
Where does shrinkage occur on microtubules?
at the plus end
old dimers can be lost here
T or F: only growth occurs at the + end of microtubules
false, both growth and shrinkage occurs here
T or F: shrinkage of microtubules occurs at the - end
false! most of the growth and shrinkage occurs at the + end
What is added to the microtubule to extend it?
new heterodimers at the plus end
Which subunit dictates assembly and disassembly of the microtubule?
beta subunit
When will microtubule assembly happen?
when the exposed B subunit at the plus end is bound to GTP
What causes disassembly at the + end of microtubules?
the GTP bound to the beta subunit is hydrolyzed and GDP + Pi cause the disassembly at the plus end
What prevents disassembly at the plus end of microtubules?
the presence of GTP-containing Beta subunits at the + end
What causes the microtubule to shrink?
if GTP hydrolysis is occurring faster than subunit addition, the GTP cap will be lost and the disassembly will outweigh the assembly
Describe a catastrophe and what causes it
microtubule disassembly from the plus end caused by GTP hydrolysis occurring more rapidly than the addition of heterodimers (the loss of the GTP cap)
Describe a rescue and how it occurs
A rescue is when enough GTP-bound tubulin dimers are added to the shrinking end of a microtubule (GTP cap) and growth can resume
Describe dynamic instability
a microtubule’s alternation between growth, catastrophe, rescue, catastrophe, etc.
What is the function of Microtubule Accessory Proteins (MAPs)?
they stabilize microtubules and promote growth/assembly
How do MAPs stabilize microtubules?
one of their domains binds to the microtubule and the other projects outwards
What mediates the binding of MAPs to a microtubule?
phosphorylation and dephosphorylation
What is an example of a MAP in humans? Describe its relationship to Alzheimer’s Disease
Tau is a MAP that is excessively phosphorylated and causes Alzheimer’s Disease
Excess phosphorylation = cannot bind to neuronal microtubules and therefore cannot stabilize the dynamic instability of microtubules
neuronal microtubules disassemble +
hyper-phosphorylated Tau aggregates = neurons die
What does the initial formation of a microtubule require?
A very high concentration of tubulin dimers and assistance of other proteins
Where are microtubules generated in the cell?
Microtubule Organizing Centres (MTOCs)
Give an example of an animal MTOC
the centrosome
Where do microtubules form in plants and fungi?
MTOCs embedded in the nuclear envelope
What does MTOC stand for?
Microtubule Organizing Centre
Describe the structure of centrosomes
two perpendicular barrel-shaped centrioles are surrounding by a dense mass (looks like a cloud) of insoluble proteins called pericentriolar material
In animals, how do microtubules anchor to the centrosome as an MTOC?
the minus end of the microtubule anchors in the dense mass of insoluble proteins surrounding the centrioles
Where are microtubules nucleated?
at their minus ends
What allows the centrosome to position itself roughly in the centre of the cell?
the position of microtubules
microtubules are nucleated (attached to the centrosome) at their minus end
the + end points outwards into the rest of the cell
What is found at the base of the minus end of microtubules?
gamma tubulin rings
What is the function of gamma tubulin rings in microtubules?
they are required for growth and polarity determination in all organisms
they also anchor the minus end of microtubules to insoluble proteins of the pericentriolar material in animal centrosomes
How do gamma tubulin rings help anchor the microtubule to centrosomes?
they are located at the base of the minus end of microtubules and bind to the insoluble proteins of the pericentriolar material (the dense mass that surrounds the centrioles) of animal centrosomes
What binds to the gamma tubulin ring once it is bound to the periocentriolar material of centrosomes? What does this cause?
the alpha tubulin of a heterodimer tubulin
this causes the elongation of the microtubule with the plus end facing outwards
What are the 2 kinds of microtubule motor proteins?
kinesins
dyneins
What is the general function of the microtubule motor proteins?
they generate the force necessary for moving materials within the cell along microtubules
What kind of material do microtubule motor proteins move?
organelles
incoming secretory vesicles
outgoing endocytic vesicles
vesicles between the ER and Golgi (COPI and COPII)
What direction do kinesins move?
toward the + end of the microtubule
What direction do dyneins move?
towards the minus end of the microtubule
What structure does a kinesin have?
a tetramer with 2 heavy and 2 light chains
Describe the structure of kinesin
a tetramer (2 heavy + 2 light chains)
globular HEADS on the heavy chain
heads are connected to the tail by a neck and flexible stalk
What is the function of the heads of kinesins?
they bind and hydrolyze ATP to create processive movement along the microtubule
What is the function of the tails of kinesins?
the tail binds the cargo that the kinesin is moving
What connects the heads and tail of kinesins?
a neck and flexible stalk
Describe what it means for the movement of kinesin to be processive
one alternating head is attached to the microtubule at all times (like walking - one head at a time)
Describe the position of a kinesins 2 heads (rear and leading) at any given time
the rear head lags behind the leader head and is bound to ATP and the microtubule
the leader head is ADP-bound and loosely connected to the microtubule
What is the first step in a kinesin moving forward one step?
the rear head hydrolyzes the ATP it is bound to (now bound to ADP)
this releases phosphate which loosens its attachment to the microtubule
What happens after the rear head of kinesin hydrolyzes ATP (Step 2)?
the front head replaces the ADP it is bound to with ATP
What happens after the leading head of kinesin replaces ADP with ATP (Step 3)?
When ATP binds to the front head it causes a conformational change which propels the rear head forwards (becomes the new leading head)
What happens after the rear head is propelled forward (Step 4)?
the new rear head hydrolyzes ATP to release phosphate and loosen its bind to the microtubule
and
the new leader head will exchange ADP to ATP to take another step forward
Briefly describe all the steps for kinesin to move forward one step
- 1 ADP bound kinesin head binds to a microtubule binding site
- this head exchanges ADP for ATP
- binding of ATP causes conformational change which propels the 2nd head in front to a new binding site
- the new rear head hydrolyzes its ATP to release phosphate and loosen its attachment to the MT
- new leader head exchanges ADP with ATP to take another step
repeat repeat
Describe the structure of dyneins
have both heavy and light chain
2 binding sites for microtubules on globular heads on heavy chains
long stem domains connect heavy and light chains
light chains bind to dynactin
What is required for dyneins to bind cargo?
the light chain of dyneins binds to a protein called dynactin which can bind to cargo
Describe dynactin?
a linker protein that binds to the light chains of dyneins and the cargo
What direction is anterograde transport?
forward
What direction is retrograde transport?
backward
In axoplasmic transport along an axon in nerve cells, where are materials moved in anterograde transport?
away from the cell body
In axoplasmic transport along an axon in nerve cells, where are materials moved in retrograde transport?
toward the cell body
What microtubule motor proteins would be involved in axoplasmic anterograde transport?
kinesins move cargo from the minus to the plus end (forward = anterograde = from cell body to axon terminus)
What microtubule motor proteins would be involved in axoplasmic retrograde transport?
dyneins move cargo from plus to minus end (backwards = retrograde = from axon terminus to cell body)
What structure do intermediate filaments have?
they are tetramers
How does the structure of intermediate filaments form?
globular terminal domains (N and C termini) for attachment between a long alpha helical region
parallel monomers associate into dimers
dimers are assembled antiparallel and staggered into tetramers to make ONE intermediate filament
T or F: all intermediate filaments are the same size and composed of the same number of tetramers
false! they can vary
What makes intermediate filaments different from MTs and MFs?
IFs do not:
have plus or minus ends (no polarity)
bind NTPs
Where are new units added to intermediate filaments?
in the middle of the filament
What is the assembly/disassembly of IFs regulated by?
phosphorylation
What makes IFs different from MTs and MFs?
they do not:
have polarity (no + or - ends) - addition is in the middle of the filament
bind NTP
break under a lot of deforming force
Order the cytoskeletal elements in ability to withstand deforming force
strongest: IF
middle: MF
weakest: MT
What is the major function of intermediate filaments?
they provide mechanical support for cells subject to mechanical stress
Where would you expect to see many intermediate filaments?
in cells that are subjected to a lot of mechanical stress
ex. epithelial cells that line the bladder
What is another function of intermediate filaments?
in addition to providing strength, they connect other parts of the cytoskeleton together
What is an example of protein in an intermediate filament?
keratin
T or F: microfilaments are less dynamic than microtubules
false! MFs are more dynamic
What are the main functions of microfilaments?
structure and stability for the cell
What are 6 examples of microfilament movements?
movement of cells over substratum (crawling)
leading edge of axon growing towards synaptic target (axon outgrowth)
organelle movements and vesicle movements
cell division (cytokinesis)
cytoplasmic streaming
muscle contraction
What are microfilaments composed of?
globular actin subunits (G-actin)
What is G-actin?
globular actin subunit (monomer) that are incorporated into the filament (F-actin)
How does G-actin assemble into actin filaments?
G-actin binds ATP and assembles into actin filaments (F-actin)
What is F-actin?
filamentous actin
What are the 2 structures of actin which make up microfilaments?
G-actin (globular) = monomer
F-actin (filamentous) = polymer of many G-actins
How is an individual G-actin monomer incorporated into an F-actin polymer?
by hydrolyzing ATP
What are the + and - ends of F-actin based on?
the shape of the monomers
How are monomers positioned in the filament? (microfilament)
positioned so the ATP binding sites are closer to the minus end
Does the + or - end of F-actin grow more rapidly?
plus
Where does disassembly occur in microfilaments?
at the minus end of the F-actin
Which part of the microfilament is weaker?
the part where the actin is bound to ADP is weaker (the minus end)
Which end of an F-actin filament has ADP bound?
minus
Which end of an F-actin filament has ATP bound?
plus end
What promotes disassembly/depolymerization in F-actin?
Hydrolysis of ATP
Describe treadmilling in microfilaments
when the F-actin polymer is adding units at the same rate the minus end is removing units
it’s like an equilibrium state and the length of the polymer is stable
What happens to the length of the F-actin polymer when treadmilling occurs?
the length is stable
What happens to individual units that have been added to the plus end during treadmilling?
they will move toward the minus end
roughly how much of a cells total protein is actin? How much of this is assembled into filaments? What happens to the rest?
5% is actin
50% of this is assembled into filaments
the other 50% remains as soluble monomers
What binds to either F- or G- actin? What does it do?
Actin Binding Proteins that modify the properties of the actin
What are 6 examples of how Actin Binding Proteins can modify F- or G-actin?
nucleate or sequester G-actin monomers
polymerize or depolymerize F-actin filaments
cap filaments to restrict length
cross-link to create branches and bundling to increase strength
sever filaments
attach actin to organelles or cell surface by membrane binding
What are 4 examples of the many different proteins that interact with actin (Actin Binding Proteins)?
thymosin
Arp2/3 complex
profilin
cofilin
How are microfilaments involved in cell movement?
the disassembly or reassembly of actin filaments can result in the movement towards site of reassembly
What are 3 examples of activities that require cell locomotion?
wound healing
development of axons
formation of blood vessels
Describe a lamellipodium
a broad, flattened area where the cell is reaching towards a stimulus via polymerization
What can a lamellipodium also be called?
a leading edge
Describe the structure of a lamellipodium
broad, very flat, fan-shaped with a ruffled edge that contains actin meshwork
Describe filopodia
the individual projections (ruffled edges of the leading edge) within the lamellipodium that contains a core of long bundled actin filaments
What is in the filopodia?
a core of long bundled actin filaments
What is located near the tail of a cell with a lamellipodium?
stress fibres
What are stress fibers? what do they do? where are they located?
contractile non-muscle actin bundles that contract near the tail of a cell to bring the rest of the cell forwards as the lamellipodium crawls toward a stimulus
What is the movement of a cell towards a stimulus called?
chemotaxis
Briefly describe the steps of cell movement with actin filaments
- actin polymerization pushes out lamellipodium
- new attachments to substratum via integrins
- stress fibres contact and cell moves
Where in a cell is actin more concentrated?
the leading edge
Where in a cell is myosin more concentrated?
at the tail of the cell
What is actin growth at the lamellipodia regulated by?
WASP proteins
What causes new filaments to form at the lamellipodia?
WASP activates Arp 2/3 complex in response to chemotactic signals
What does Arp 2/3 stand for?
Actin Related Protein
What occurs in individuals with Wiskott-Aldrich Syndrome?
they are lacking WASP proteins and have a dysfunctional immune system
white blood cells fail to respond to chemotactic signals
What activates ARP2/3?
WASP
What happens when ARP2/3 has been activated?
ARP2/3 will bind to the minus end of an actin filament to promote nucleation
What happens ARP2/3 binds to the minus end of an actin filament?
it can attach to the side of another actin filament to form a branch
What is the purpose of ARP2/3 forming branches?
it helps individual actin filaments extend faster and extend into a tree-like web
What does cofilin do to microfilaments?
it is an actin binding protein that encourages the minus ends to dissociate
What does profilin do to microfilaments?
it is an actin binding protein that encourages the growth at plus ends
T or F: actin can form fixed permanent cell projections
true
What kind of micrograph would be used to visualized microfilaments?
TEM
What is an example of microfilaments that would require staying the same length?
actin filaments in microvilli
Describe the structure of microvilli
plus ends of actin filaments form microvillii and are bound to the tip of the plasma membrane by minus ends anchored to intermediate filaments
stabilizing proteins run along the length of the microvilli to maintain the length
What stabilizes the length of microvilli?
stabilizing proteins
What type of motor proteins are involved with microfilaments?
myosin
What are the 2 types of myosin?
conventional (type II)
nonconventional
Describe the function of conventional (type II) myosins
they are used for contraction in actin filaments
Describe the function of nonconventional myosins
used like kinesins to move things around the cells (like vesicles)
What does myosin use to move things?
ATP hydrolysis
Describe the structure of myosin II
a dimer made up of monomers with
1 heavy chain and 2 light chains
2 globular head domains
a coiled coil tail of heavy chain alpha helices (C terminal chains)
In myosin, what hydrolyzes ATP?
the two globular head domains bind and hydrolyze ATP (N terminus)
What can type II myosins assemble into?
bipolar filaments (many myosins) with heads in both directions
What causes the contractile force of type II myosin?
the pulling by bipolar filaments on the actin cytoskeleton
