Lecture 13- cytoskeleton 2 microtubules, cilia and flagella

Intermediate filaments

Microtubules

Microfilaments

what proteins use energy to move directionally
along cytoskeletal polymers

motor

moves along Tub to neg end

Dynein

moves along Tub to pos end

Kinesin

moves along Actin

Myosin

what contribute to structural support, organization, motility within the cell

microtubules

microtubules are what

polymers

microtubules made up of α and β subunits that can be added and released

heterodimers

# protofilaments

13

what is stabilized by associated proteins

microtubules

subunits are rapidly incorporated as dimers with what-bound

to the β-subunit, then what is hydrolyzed

GTP

what on β is exchanged in the cytosol for what

GDP, GTP

microtubule-organizing centers (MTOC) govern microtubule what

assembly

microtubule- organizing centers control centrosome and basal

body in what cells

animal

microtubule- organizing centers control nucleus in what cells

plant

centrosome is made up of # centrioles and PCM

(pericentriolar material)

2

microtubule assembly: dimers are added to (and lost from) which end

pos

𝛾-Tubulin sits in a ring in PCM and what bind to it to form microtubule

dimers

minus end is associated with what

centrosome

if all GTP is hydrolyzed, the microtubule can rapidly what

depolymerize

microtubule assembly & disassembly: requires energy from
GTP hydrolysis (beta) to what (expensive)

lengthen

some treatments change microtubule dynamics: binds dimer, adds to tubule, blocks further addition

colchicine

some treatments change microtubule dynamics: suppress addition
to tubule, may detach from MTOC

nocodazole,vinblastine

some treatments change microtubule dynamics: inhibits disassembly and thereby assembly

taxol

taxol, nocodazole,vinblastine, colchicine are ANTI CANCER or CANCER agents because they interfere with cell division

anti cancer

dynamic microtubule structures are regulated in cells
by

Microtubule Associated Proteins (MAPs)

EB1 binds near the what of microtubules

end

you attempt to make tubulin polymers in vitro
you add cell extracts to large amounts of 𝛼- and 𝛽-Tubulin,
ATP, GTP, GDP, and adjust the temperature to 37

after you get polymers, which of the following will be depleted
from the test tube?

GTP

you attempt to make tubulin polymers in vitro
you add cell extracts to large amounts of 𝛼- and 𝛽-Tubulin,
ATP, GTP, GDP, and adjust the temperature to 37

you have made tubulin polymers in vitro, but they are transient, which is not great for taking pictures.
which drug might help you maintain the microtubules

taxol

About your in vitro microtubules…
after much work (EM), you realize your polymers are
funny-looking because the tubes are too small in
diameter

which of these additions would help?

centrosomes = 𝛾-Tubulin ring complex

Taxol-treated cells have more or less dynamic microtubules

less

two motor protein families

use energy to move in opposite directions along microtubules

Dynein & Kinesin

•first implicated in cilia and flagella movement; later found in all animal cells (cytoplasmic)
•stalk binds MTs; head generates force; requires ATP
•binds cargo via dynactin protein
•moves organelles, vesicles, particles; positions Golgi,
centrosomes, mitotic spindle (required for cell division)

Dynein

~ 45 different blank-like proteins (KLPs) in mammals
• heads “step” along protofilament, 1 tubulin subunit at a time towards + end
•[ATP] dependent
•processive
• tend to move organelles (mitochondria, peroxisomes) and vesicles outward

Kinesin

Can Kinesin work upside down?

Yes

how fast (and expensive) is Kinesin movement?
one step is about 8nm
let’s say the distance from Golgi to the plasma membrane of an epithelial cell is 16 micro meters, and that kinesin can carry cargo there in 16 seconds. Each step expends 1 ATP
How many ATPs does this trip use?

2x103

in cultured neurons, your GFP fusion motor protein tends to move toward the cell body. is your motor protein a plus-end or minus-end directed motor?

minus

In cultured neurons, your GFP fusion motor protein
tends to move toward the cell body.

what known kind of motor protein does this resemble?

Dynein

what are microtubule-rich structures that beat or wave

flagella

protozoans, algae, sperm use flagella for what

movement

what are microtubule rich structures that can wave or be stationary

cilia

The axoneme of cilia/flagella is made up of microtubules and what

dynein

what proteins are also needed to build/maintain
the axoneme of cilia/flagella

motor

Kinesin 2 and what dynein transport cargo in and out

cytoplasmic

cilia move by microtubule sliding via ciliary what

Dynein

a mutation that disrupts which of the following proteins is the most likely to result in this outcome: Kartagener Syndrome
cilia exist but have defective or no movement

ciliary dynein

Kartagener Syndrome: cilia exist but have defective or no movement

you want a mouse model of the disease, so you want to make a genetic mutant. you know that the human patients have only small changes in the coding region of the dynein protein, not entire loss, and you know that cilia from these patients hydrolyze ATP at a similar rate to normal cilia.

so, which region of the protein(s) might be the best place to make a small deletion to mimic the disease?

heavy chain stem domain