Module 7 - The Cytoskeleton Flashcards
1
Q
Cytoskeleton
A
provides cells shape and structure
- necessary for the shape of specialized structures in differentiated cells such as microtubules in cilia and actin in microvilli of epithelial cells
- cell shapes depends upon the unique functions of different filaments, and so in neurons we see microtubules providing structures to the elongated axons, while actin provides shape and function to the growth cone
- cytoskeleton must also. be dynamic to provide movement such as migration of cells and cell division
2
Q
3 classes of filaments in eukaryotic cells
A
- defined by their diameter and type of subunit used as building blocks
1) actin filaments = typically labelled using fluorescently tagged phalloiding molecule, phalloiding binds to actin monomers with a high affinity and high specificity, but it also stabilizes the filament when bound. Actin can also be labelled with an antibody to actin or with a protein fusion such as actin GFP
2) microtubules = labelled using antibodies specific to one of the tubulin subunits or using a protein fusion such as tubulin GFP
3) intermediate filaments = there are many types of IFs that can be labelled using an antibody specific to a monomeric subunit of the filament or a GFP fusion protein
3
Q
Filament composition
A
each filament is constructed from smaller protein subunits to form a long polymer
1) actin = thinnest filament and are composed of monomeric actin subunits
2) microtubules = the thickest filament and are made up of dimeric subunits of alpha and beta-tubulin
3) many intermediate filaments each is assembled. from a different protein or set of proteins
4
Q
filament distribution
A
3 filaments are distributed differently in each type of cell
in epithelial cells:
1) actin forms the shape of the microvilli at the apical cell surface
2) intermediate filaments span the cell to provide structural support. ones made with lamin proteins form the nuclear lamina that provides structure and shape to the nucleus
3) microtubules form networks for intracellular transport
5
Q
motor proteins
A
there are filament specific motor proteins that track along actin filaments and microtubules
- myosin proteins move along actin filaments
- kinesin and dynein track along microtubules
- general structure: head domain binds to a cytoskeletal fiber and the tail domain attaches to a cargo
- ATP hydrolysis provides energy for walking
6
Q
Actin based structures and movement
A
- highest density of actin is at the cell periphery
- actin filaments underlying the cell membrane determine the shape and movement of the cell surface
- typical actin based functions include the establishment of microvilli, the formation of contractile bundles that form the sarcomeres that power muscle cell contraction
- formation of filipodia and lamellipodia needed for cell migration and contractile ring that directs cytokinesis
7
Q
actin filament structure
A
are 2 stranded helical polymers with each polymer build from actin monomers called G-actin
8
Q
actin filament polarity
A
actin filaments are polar, 2 ends look and behave differently from one another
- myosin head protein binds in just one orientation on the actin filament
- under the EM can see the proteins point away from the actin filament
- defines a plus and minus end based on the rate of actin polymerization
- (+) end grows more quickly through the. addition of more actin subunits and has a barbed appearance (chevron end of arrow)
- (-) end grows more slowly and may shrink and has a pointed appearance
9
Q
G-actin monomer
A
a single actin monomer globular actin (G-actin) is divided into 4 structural domains with a large cleft between domains 2 and 4
- cleft forms an ATP nucleotide binding site
- each actin monomer is polar, thus the microfilaments built up from these subunits are also polar
- ATP binding pocked is pointed to the minus end of. the elongating polymer, so that the ATP binding pocket of each monomer is not exposed within a filament except for a pair of monomers right at the minus end
10
Q
actin polymerization and depolymerization
A
- an actin filament is not a static structure
- it is dynamic, constantly engaging in polymerization and depolymerization
- can occur at plus and minus end, but tends to be more growth at plus and more shrinkage at minus-end
- ATP binding regulates the growth and disassembly of the actin filaments
- Actin ATP monomers in the cytosol can join the plus end as long as the [ ] of actin ATP is high enough
- actin as intrinsic ATPase activity that hydrolyzed ATP to ADP and replaces inorganic phosphate (Pi)
- this happens within the polymer so that most of the actin filament is made up of actin-ADP
- ADP is not released because the nucleotide-binding site is covered in the actin filament
- the rate of polymerization is greater than the rate of depolymerization at the (+) end where actin-ATP monomers are added
- Actin-ADP tends to come off the (-) end readily where the rate of depolymerization is greater than the rate of polymerization
- in the cytosol, free actin-ADP can release ADP and exchange it for ATP
11
Q
F-actin
A
-filamentous actin (F-actin) is created through the polymerization of actin monomers
12
Q
critical concentration
A
concentration. at which the rate of actin monomer addition is equal to the rate of removal, means no net growth at that end
- if the [ ] of actin monomers is greater than the critical [ ], then the rate of polymerization will exceed the rate of depolymerization and the end of the filament will grow
- if the [ ] of actin monomers is lower than the critical [ ], then the rate of depolymerization will exceed the rate of polymerization, and the end of the filament will shrink
- the critical [ ] and the working [ ] of actin monomers are different at each of the ends so that different dynamics can be seen at each end
13
Q
factors regulating the rates of actin dynamics in the cell
A
- prolifin: binds to actin-ATP promoting ATP binding and activating the monomer. The prolific actin dimers accumulate at the plus end, increasing the local [ ] of active actin monomers
- thymosin: binds to actin monomers and inhibits polymerization, however thymosin actin dimers also accumulate at the plus end, creating a buffer of stored actin monomers
- capping proteins: at the ends of actin filaments can inhibit polymerization or depolymerization
14
Q
Treadmilling
A
- a phenomenon observed with actin filaments in which there is no net increase in the length of the filament because the rate of polymerization at the (+) end is equal to the rate of depolymerization at the (-) end
- while there is. no increase in the length of the filament, the relative position of the filament in the cell is changing, and effectively the filament is moving forward
15
Q
actin and cell migration
A
actin filaments can power the movement of cells through the reorganization of actin filaments that push out the cell membrane
-thisis observed through formation of filipodia and lamellipodia in a migrating cell
16
Q
Myosin: AF-based motor
A
myosin motor proteins area able to move along actin filaments and power intracellular cargo trafficking
- 8 identified family members
- myosin I, II, and V are present in nearly all eukaryotic cells and distinguished by structure and function
- all share a characteristic motor (head) domain at the N-terminus which has a site that binds actin filaments and a site that binds and hydrolyzed ATP to drive the motor
- while head domains of myosin are similar, tail domains are highly divergent in order to carry different cargo at different rates
- most myosin moves towards the plus. end of actin filaments
17
Q
myosin II
A
contains 2 heavy chains that form a coiled coil motif and 4 light chains of 2 distinct types
-the motor domain heads can be seen as small globular domains at the top of the tails
18
Q
regulation of myosin assembly
A
phosphorylation of myosin light chains of myosin II by a MLC kinase drives polymerization of the myosin proteins by initiating extension of the myosin tails and activating the actin binding domain on the motor heads
19
Q
myosin II bipolar thick filament
A
- an assembly of 15-20 myosin II proteins forms a bipolar filament called myosin II thick filament
- bipolar filament has myosin motor heads to the left and right of a bare patch of myosin tails
- the motor heads are exposed for association with actin filaments
20
Q
contractile muscles
A
- contain sarcomeres
- within the multinucleated contractile skeletal muscle cells, the myosin II thick filaments are associated with actin filaments (thin filaments) into a structure called a sarcomere
- the term “striated muscle” comes from the appearance of these structures
- the plus end of the actin filaments are fixed to Z-discs within the sarcomere
- capping proteins cap the ends, tropomodulin at the minus end and capZ at the plus end
- nebulin binds together parallel actin filaments
- the myosin thick filament are in the middle, they are also attached to the Z-discs but by a giant molecular spring called the titin protein
21
Q
sarcomere dynamics
A
- the actin filaments are pulled past the myosin filaments towards the middle of the sarcomere by the cyclical association with the myosin motor heads
- this. causes shortening of the sarcomere without any change in thick or thin filament length
- each myosin head cycles through ATP binding and ATP hydrolysis to ADP in order to track along the actin
- this causes shortening of the sarcomere and muscle contraction
- when the myosin thick filament releases the actin thin filaments upon the dissociation of calcium from the actin filaments, they slide past one another to allow sarcomere elongation and muscle relaxation
22
Q
actin and myosin cycle
A
- conversion of chemical to mechanical energy is mediated my myosin
- myosin proteins undergo series of conformational changes regulated by ATP binding and hydrolysis
1) attached (myosin): myosin is attached to actin
2) released (myosin-ATP): ATP binding to myosin releases actin
3) relaxed (myosin-ADP and Pi): ATP is hydrolyzed to ADP and Pi, changes myosin conformation returning it to the relaxed conformation
4) attachment (myosin-ADP, release of Pi): the release of inorganic phosphate increases the affinity of the myosin head for actin and allows binding
5) powerstroke (myosin, release ADP): the release of ADP. changes myosin conformation again. since the myosin is attached to actin, this. pulls the actin filament, this puts the cycle back at step one. ATP binding releases myosin from actin again. - This cycle is repeated many times during muscle contraction.
- binding and hydrolysis of the ATP molecules moves the motor a few nanometers along the actin tract
23
Q
myosin V moving melanin
A
powers intracellular trafficking of cargo along actin filaments
- example of this is the movement of melanosomes in skills cells called melanocytes
- melanosomes are membrane enclosed organelles containing pigment granules called melanin
- each melanocyte in the epidermis has several dendrites that stretch out to connect it with many keratinocytes
- incorporation of melanin into the keratinocytes and distribution at the apical periphery of the cells, protects the cells DNA from UV damage in a process called tanning
- while microtubules are also involved in transport of melanosomes, myosin V distributes the melanosomes to the cell membrane along actin filaments
24
Q
loss of function mutation in myosin V
A
leads to a phenotype called the dilute phenotype in which pigments associated with fur colour are not distributed into the fur and the resulting colour is diluted
25
rate of myosin motor protein movement
varies with different myosin proteins and can range from 0.2 to 60 um/sec
- the rate depends upon the cycle of nucleotide binding and hydrolysis and varies with
1) the rate of ATP hydrolysis by ATPase
2) the proportion of time myosin is bound to the actin filament, a result of affinity
- myosin V spends up to 90% of the cycle bound to actin vs 5% for myosin II which slows the rate of myosin V movement
26
factors affecting step size
myosin step size is dependent on its lever arm length, as this is the distance by which the powerstroke propels the myosin forward
- myosin V lever is 3x longer than that of myosin II
- myosin V steps in a hand over hand fashion: the trailing head detaches from actin and is propelled towards the barbed end during the powerstroke of the leading head
- the trailing head now becomes the new leading head
27
microtubules
tube comprised of 13 protofilaments arrayed in a circular pattern to form a very strong wall
- the basic subunits of the filaments are dimers of alpha and beta tubulin proteins
- the protofilaments are staggered, so that if you follow a string of tubulin monomers, they may appear to spiral up through the microtubule like a spring
28
alpha and beta tubulin binding
both alpha and beta monomer subunits are bound to GTP
| -the alpha subunit is tightly bound to GTP while the beta subunit is less tightly bound
29
hydrolysis of alpha and beta tubulin
- the GTP bound to alpha subunit is never hydrolyzed and does not exchange with nucleotides in solution
- the GTP bounds to beta subunit is cyclically hydrolyzed to GDP and the GDP is exchanged for GTP
30
alpha and beta affinity
- alpha and beta subunits are added and removed as dimers to the filament
- alpha-beta-GTP has a might higher affinity for the microtubule than alpha-beta-GDP
31
dynamics of microtubules
dimers are oriented so that the beta subunit is closer to the positive end than the alpha subunit
- dimers containing alpha-beta-GTP are added to the plus end of a growing filament, while dimers containing alpha-beta-GDP are released
- GTP hydrolysis occur within the polymerized microtubule
- most of the microtubule consists of dimers containing alpha-beta-GDP
- a GTP cap (alpha-beta-GTP) at the plus end favours growth rather than shrinkage
- alpha-beta GTP dimers have 4 times slower dissociation rate compared to the alpha-beta-GDP dimers due to the high affinity of the alpha-beta GTP for its neighbours
32
EB1-GFP
a plus-end binding protein that prevents premature catastrophic and act as a positive regulator of microtubule growth
33
dynamic instability
the (+) end of the microtubule exhibits dynamic instability, an oscillating behaviour between growth and shortening
-the [ ] of free alpha-beta-GTP dimers is maintained at a level that allows polymerization
34
MAPS
- microtubule-associated proteins
- proteins that control assembly and disassemble of microtubules
- many interconnect microtubules to help form bundles (cross-bridges) or increase stability, alter. rigidity and influence assembly rate of the microtubules
- MAPs are classified into 2 groups based on their function
1) stabilization of the filament = promotes growth, frequencies of catastrophes suppressed and/or growth rate enhanced
2) destabilization of the filament = frequency of catastrophes increased
35
microtubule nucleation
- gamma tubulin is involved in the nucleation of microtubules
- gamma-tubulin is present in much smaller amounts in cells compared to alpha and beta-tubulin
- gamma-tubulin and other associated proteins from the gamma-tubulin ring complex or y-TURC
- this ring nucleates at the minus end of a new microtubule by forming a template for the growing plus end
- gamma TURC acts as. a cap for the minus end, while microtubule grown and dynamics occur at the plus end
36
MTOC
microtubule-organizing center (MTOC) is a specific location inside the cell where microtubule nucleation occurs
- in animal cells, the MTOC is called a centrosome consists of 2 cylindrical structures called centrioles and. a cloud of pericentriolar material (PCM) that contains multiple y-TURC complexes
- the minus end of the microtubules are nucleated at the y-TURC complexes
- microtubule plus ends are directed towards the periphery of the cell
37
MTOC in mitosis
during mitosis, the microtubules of the mitotic spindle attach to the chromosomes and orchestrate replicated sister chromatids separation
- mitotic spindles are dynamic structures that are assembled and disassembled during the cell cycle
- the cycle depends upon the dynamic instability of the microtubules
- centrosomes are duplicated in mitosis to create the 2 MTOCs
- as the replicated MTOC separate, microtubules are. nucleated at the y-TURC complexes and the plus ends grow outward
- some of the microtubule plus ends emanate towards the cell periphery and anchor the spindle
- others grow towards each other to create the spindle and attach to the condensed replicated chromosome
38
microtubule reorganization during interphase
-the network of microtubules fills the cell and the nucleus is stained using a fluorescent DNA dye called DAPI
39
microtubule reorganization during metaphase
- chromosomes are compacted and associated with the bipolar mitotic spindle
- compact, replicated chromosomes are aligned at the equator of the metaphase spindle to attachments between the microtubules stretching from the poles of the spindle to attachment sites at the centromeres at the chromosomes
40
microtubule reorganization during anaphase
- the spindle poles move apart and the microtubules attach to the chromosomes shorten
- together, these mechanisms have the effect of pulling the replicated sister chromatids apart
41
colchicine
- derived from a plant, the meadow saffron or autumn crocus
- it acts as a microtubulin toxin, by inhibiting polymerization
- binds and stabilizes free alpha-beta tubulin dimers
- while the bound dimers can be incorporated into the growing microtubule, they prevent the subsequent addition or loss of other tubulin subunits
- cells in mitosis treated with colchicine will arrest in metaphase without chromatid separation
42
Taxol
- binds to beta-tubulin and increases the affinity of the dimer to the (+) end
- the effect of this is to prevent the assembly of the mitotic spindle and thus inhibit mitosis
- for this reason, taxol has been used as an. effective cancer treatment where it is commonly referred to as the patented drug "paclitaxel"
- it is derived from the pacific yew tree (taxis breviofola) and has proven difficult to synthesize in the lab
- 2 other chemical cause rapid depolarization of microtubules --> vinblastine and nocodazole
43
microtubules motors direct intracellular traffic
kinesin (move towards the plus end)
| dynein (move towards the minus end)
44
kinesin
- a tetrameric complex comprised of 2. identical heavy chains and 2 identical light chains
- the globular heads at the N-termini of the heavy chains are called the motor domains
- these cyclically bind to microtubules and generate movement through ATP hydrolysis
- kinesins move cargo including vesicles and organelles towards the (+) ends of the microtubule, thus they generally move cargo the periphery of the cell
- while the motor proteins are highly conserved across family members, the tails are highly variable
- it is the tails that determine the specificity of cargo binding
45
the mechanochemical cycle of kinesin
described as "hand-over-hand" motion
- there are 2 motor domains in the kinesin dimer and one is always attached to the microtubule
- start at the point in the cycle where the lagging head is. bound to ATP and the leading heads is bound to ADP.
- ATP kinesin has a higher affinity for the microtubule than ADP kinesin
- in the lagging head, the ATPase motorhead hydrolyzes ATP to ADP, this reduces the affinity of the head for the microtubule
- in the leading head, ADP is exchanged for ATP, this increases the affinity of the head for the microtubule
- binding of ATP induces a conformational change in the neck region that causes the lagging head to swing forward in front of the leading head towards another microtubule-binding site, resetting the cycle back to the top
46
bead assay
under normarski microscope, researchers were following plastic beads tethered to kinesin
-the bead is one micron size and attached to kinesin that is moving along a microtubule track made from purified tubulin
47
gliding motility assay
under fluorescent microscope, kinesin proteins are tethered to a glass slide at their tail (cargo) ends
-the motor proteins are then able to move short, fluorescently labelled microtubules in solution above the slide
48
dynein
- minus end-directed motor
- 2 forms
1) cytoplasmic dynein is associated with the microtubules that direct movement of organelles and vesicles in the cytoplasm
2) axonemal dynein is found in structures that power the movement of whole cells such as the cilia and flagella
- dynein contains 2 identical heavy chains and a variety of intermediate and light chains
49
dynein power stroke cycle
1) ATP binding releases the motorhead group from the microtubule
2) ATP hydrolysis, dynein-ADP + Pi can now attach to the microtubule
3) release of Pi powers the power-stroke of the linked, which pulls cargo to the left the cargo moves
movement of cargo is. driven by the Powerstroke of the linker that occurs with. release of inorganic phosphate from ADP
-within a dynein dimer, the dynein proteins alternate power-strokes to move the cargo
50
movement of vesicles
- cargo is carried by both dynein and kinesin motor proteins
- the same cargo can move back and forth along the microtubule-dependent upon the. associated motor protein
- microtubules span the long axons of neural cells
- while the (-) ends are anchored at the MTOC near the nucleus within the cell body, the (+) ends extend towards the periphery of the cell and along the axons, towards the cell membrane of the synapse
- vesicles carrying NT are carried along from the cell body to the synapse along the axons
- video of fluorescently labelled vesicles in neural axon shows that transport can occur along these microtubule highways in both directions
51
microtubule pulling of cargo
- cargo is transported along microtubules by teams of molecular motors
- these motors are pulling the cargo in opposite directions, dynein towards the minus end and kinesin towards the plus end
- one model suggests the proteins are engaged in a molecular tug of war
- the final direction of movement is decided by the winner of the battle
- there are regulatory proteins that control direction in response to signals within the cell
52
function of changing direction in melanosomes
- melanosomes are pigment filled organelles, in many species of fish, the movement of the organelles change the colour of the skin cells in response to behaviour signalling
- molecular motors carry melanosomes out to the periphery of the cell or concentrate them at the center
- the motors responsible for concentrating the melanosomes at the center are dynein which moves the melanosomes along microtubule tracts towards the minus ends at the MTOC near the center of the cell
- kinesin motors are responsible for dispersing the melanosomes
- since the plus end of the microtubules are oriented towards the periphery, kinesin will carry melanosomes to the periphery
- dispersion of melanosomes to the periphery causes the cel to appear darker
- the concentration of melanosomes towards the center will cause the cell to appear lighter colour
- signalling the alternating control of the motor proteins are signals that use cAMP as a secondary messenger in the signal transduction pathway
