Cell Bio - Exam 3

25 nm

diameter of microtubules

microtubule organizing center (MTOC)

most microtubules emanate from a ______________ ______________ _____________ (____)

tubulin

microtubules are made of _______________ monomers

1. organization of intracellular organelles and transport of vesicles (motor proteins)

2. beating of cilia and flagella

3. structural integrity of nerve cell, red blood cell, and flagellar structure

4. alignment and separation of chromosomes during mitosis and meiosis

microtubules play a role in

heterodimers, α-tubulin and β-tubulin

microtubules are made of _____________dimers that consists of a _______-__________ and _______-__________

1 α-tubulin and 1 β-tubulin

one subunit of a microtubule consists of

α-tubulin, β-tubulin

_-tubulin has permanent GTP while _-tubulin is GDP/GTP exchanged

"one strand" of repeating subunits for a microtuble

protofilament

55 kDa

what is the weight of α-tubulin and β-tubulin?

involved in nucleation of microtubules, doesn't become part of final structure

gamma tubulin

13 protofilaments, majority of microtubules in cytoplasm (11-15 protofilaments observed occasionally)

singlet

consists of an A tubule and a B tubule, cilia and flagella. 23 protofilaments

doublet

consists of an A tubule, B tubule, and C tubule. centrioles (centrosomes), basal bodies (flagella, cilia), MTOC. 33 protofilaments

triplet

1. unstable, short lived - assembles and disassembles rapidly

2. stable, long lived - remain polymerized for long time

2 populations of microtubules

need to re/depolymerize as cell needs it. example is cell division microtubules in mitotic apparatus

unstable, short lived MT

maintain structural integrity of cell. examples are sperm flagella, red blood cells (marginal band), platelets, nerve cells (axons and dendrites)

stable, long lived MT

intermediate filaments

in a nerve axon, microtubules work together with ________________ ___________________

run almost along-side MT and wrap around nuclei for integrity

in a nerve axon, intermediate filaments do what?

any structure used by cells to nucleate and organize MT (lots of different kinds)

microtubule organizing center (MTOC)

centrosome

primary MTOC in animal cells

basal body

what organizes MT in flagella?

microtubules

basal bodies and centrioles are made of ____________________

-, +

MT have a ___ end and a ___ end

polar

because of the different ends of MT, MT are said to be _________________

-

in MT emanating from centrioles, the ___ end of MT are bound to organizing center

+

in MT emanating from centrioles, the ___ end of MT are furthest from the center

inside centrosome, very short triplet MT

centriole

centrioled

the spindle poles arise by division of _______________

different

in the dendrites of nerve cells, the microtubules are in (same/different) directions

same

in the axon of nerve cells, the microtubules are in (same/different) directions

NO

do nerve cells use a MTOC?

YES - but not in same orientation

in the dendrites of nerve cells, do MT extend the whole length of the dendrite?

NO - but in same orientation (-) -> (+)

in the axon of nerve cells, do MT extend the whole length of the axon?

the cell body

in the axon of nerve cells, the (-) end is facing where?

the length of the axon

in the axon of nerve cells, the (+) end is facing where?

lower, higher

in MT, the (+) end has a (higher/lower) critical concentration while the (-) end has a (higher/lower) critical concentration

treadmilling, addition, loss

_____________________________ occurs along the length of MT due to (addition/loss) at the (+) end and (addition/loss) at the (-) end

depolymerization of MT

if a cell is in interphase, what will happen with colcemid is added or cooled to 0 degrees C?

re-polymerization of MT

what will happen is colcemid is removed or the cell is warmed to 37 degrees C?

reversible

the depolymerization/repolymerization experiment shows that this process is (permanent/reversible)

nucleate

MTOC can ______________ MT

BOTH: yes (+ and - ends)

microfilaments vs. microtubules: polar

microfilaments: Yes-ATP

microtubules: Yes-GTP

microfilaments vs. microtubules: binding nucleotide

actin-ATP (in vivo)

actin-ADP (in vitro)*

Actin-non hydrolyzable ATP analogs (in vitro)*

(**Although triphosphate nucleotides (ATP or GTP) are required for subunits to polymerize in vivo, it is possibleto force subunits bound to diphosphate nucleotides (ADP or GDP) or to non-hydrolysable analogs of ATP/GTP to polymerize in vitro (in the test tube).)

subunits that can polymerize microfilaments

tublin-GTP (in vivo)

tubulin-GDP (in vitro)*

Tubulin-non hydrolyzable GTP analogs (in vitro)*

(**Although triphosphate nucleotides (ATP or GTP) are required for subunits to polymerize in vivo, it is possibleto force subunits bound to diphosphate nucleotides (ADP or GDP) or to non-hydrolysable analogs of ATP/GTP to polymerize in vitro (in the test tube).)

subunits that can polymerize microtubules

microfilaments: ATP hydrolysis required

microtubules: GTP hydrolysis required

microfilaments vs. microtubules: depolymerization

colchicine and taxol

drugs that disrupt microtubule dynamics

- bind between α and β tubulin on dimers

- high concentration: causes depolymerization

- low concentration: stabilizes

colchicine

- binds to side of tubules

- stabilizes

- used in chemotherapy, messes up cell divison

taxol

1. microtubules-associated proteins (MAPs) (stability)

2. proteins that regulate (+) end polymerization/connect MT to other structures (+TIPS (plus-end trafficking proteins)

3. proteins that regulate (+) end depolymerization

4. motor proteins, intraflagellar transport, ciliopathies

proteins associated with microtubules

stabilize MTs. interact with side of MT => different MAPs have different arm lengths

Microtubules associated proteins (MAPs)

MAP with a long arm, has a (+) charge with binds to the (-) charge of MT

MAP2

MAP with short arm

Tau

different organization

different arm lengths of MAPs cause?

plus-end trafficking proteins

regulate polymerization at the (+) end, e.g. EB1 and EB3

+TIPs

1. promote growth

2. prevent disassembly

3. connect MTs to other structures

how do +TIPs work?

stacked images of same MT show that +TIPs associate with MT at the (+) end. can visualize growth and shrinkage

how do +TIP's like EB1 and EB3 recognize/attach to the + end

EB1 recognition of specific plus end structure allows it to bind, other +TIPs hitchhike on EB1. could be other proteins that interact as well

proposed mechanism of +TIPS

sometimes rapid reorganization requires loss at both ends, so loss at (+) end occurs too

proteins: kinesin-13 and stathmin

proteins that regulate microtubule disassembly at (+) end

binds to (+) end, uses ATP hydrolysis to bend MT. destabilizes MT so they fall off more quickly

end disassembly, bend MT to promote disassembly

kinesin-13

enhances intrinsic GTP hydrolysis by increasing activity of B4

stathmin

sufficient, necessary

MTOC are ______________ to nucleate MT growth, but they are not _________________

kinesin physically bends microtubules by creating a convex shape, which creates force to bend MT

how does kinesin-13 bend MT?

kinesins (cytosolic) and dyneins (cytosolic + axonemal)

two families of microtubule-based motor proteins

+

Kinesins move towards the _____ end of the microtubule.

-

dyneins move towards the _____ end of the microtubule.

- globular heads (bind to strand)

- 2 heavy chains where ATP hydrolysis occurs, binding to MT

- coiled-coil α helix

- 2 light chains that bind to transported vesicles, help with cargo and motor connection

EXACTLY LIKE MYOSIN

cytosolic kinesin structure

organelle transport

hand-over-hand stepping

Kinesin-1 (conventional) and Kinesin-2 (heterotrimeric)

globular heads at both ends

important in organelle organization

sliding (slide MT in relation to each other)

kinesin-5 (bipolar)

hydrolysis of ATP into ADP promotes stepping mechanism

transport of vesicles by cytosolic kinesins

1. leading heads bind ATP

2. lifts off and swings trailing head forward

3. new leading head binds

4. hydrolysis of ATP of old leading end, ATP binding at new leading end

(see image)

motor activity of cytosolic kinesins

- stem with dynactin binding domain (interacts with cargo)

- head with ATPase domain

- stalk with mictotubule binding domain

structure of cytosolic dynein

move towards (-) end of ginormous proteins (4000 AA0

rotates, which moves MT-binding domain towards (-) end

motor activity of cytosolic dynein

kinesin, dynein

_______________ move cargo away from MTOC while ______________ move cargo towards MTOC

actin, microtuble

cooperation between ________-based and _______-based motor proteins allows the movement of vesicles through the cell

- 1/2 per cell

- locomotion (sperm, single-cell eukaryotes)

flagella

- many per cell (all around cell)

- locomotion (single cell eukaryotes)

- moving particles or fluid over cell surface (tracheal cells)

cilia

"9+2" arrangement = 9 doublets (A and B subunit) and 2 singlets

structure of axonemes of cilia or flagella

outer dynein arm

What is this?

what is this?

outer dynein arm

what is this?

inner dynein arm

what is this?

plasma membrane

what is this?

nexin

what is this?

bridge connecting central singlets

what is this?

central pair of singlet MT

what is this?

inner sheath

what is this?

B tubule

what is this?

A tubule

what is this?

doublet MT

what is this?

spokehead

what is this?

radial spoke

A tubule has how many protofilaments?

13

B tubule has how many protofilaments?

10

A protein that links microtubule doublets to each other in the axoneme, important in flagellal beating

nexin

-

the inner and outer dynein arms move toward the ___ end

radial spoke

inner sheath

what parts of the axoneme are important in structural integrity?

1 base

3 heads

large

structure of axonemal dynein

base has stable association with A tubule

head has transient interactions with B tubule using ATP hydrolysis

base binds with A tubule while head walks along neighboring B tubule (not same A and B pair)

how does axonemal dynein interact with A and B tubules?

nexin connects tubulin pairs, so when the tubule tries to move (because of dynein activation) it can't. results in bending, which is progated throughout flagella and creates a wave

dynein sliding of axonemal microtubule doublets

no nexin => no sliding

activation of dynein causes MT to slide past one another as they walk towards (-) end. think of it as people "dragging" tubule

dynein sliding of axonemal microtubule doublets when nexin linkers removed by protease