Douglass

Question 1

OPTICAL BIREFRINGENCE

Answer 1

Birefringent materials have different indices
of refraction for light polarized parallel or
perpendicular to the optical axis.
Two beams with orthogonal polarization
are produced if illumination is at an angle
to optical axis

Question 2

SHINYA INOUE AND THE DISCOVERY OF MICROTUBULES

Answer 2

Two assumptions:
(1) Cellular birefringent structures
might be important.
(2) We might learn something by
watching them change over time.

saw mitotic spindles

Question 3

______ DISRUPTS THE MITOTIC SPINDLE

Answer 3

colchicine

Question 4

COLCHICINE AND THE IDENTIFICATION OF TUBULIN

Answer 4

Gary Borisy and Ed Taylor (1967).
Treated sea urchin eggs with 3H-colchicine.
Lysed and fractionated by chromatography
and sedimentation.
Tracked the 3H-colchicine and found it bound
to a single protein.

Question 5

Tubulin dimers

Answer 5

heterodimers
Two isoforms of tubulin: αand β
50% sequence identity
Typically exist as a heterodimer:
Kd = 84 nM
Bind to and hydrolyze GTP

Question 6

Microtubule structure

Answer 6

α/β-tubulin heterodimers incorporate into
protofilaments
Thirteen protofilaments associate laterally
to form a microtubule
Diameter = 25 nm
Stereotyped orientation of heterodimers
creates a polarized filament
MTs are very rigid

number of protofilaments/alignment can vary

Question 7

CENTROSOME

Answer 7

organizes microtubules
All cells contain a centrosome (aka microtubule
organizing center, MTOC), defined by two
centrioles surrounded by insoluble pericentriolar
material (PCM)
One centrosome per interphase cell
In mitotic cells, most microtubules are anchored
to the centrosome by their minus ends
Creates a stereotyped orientation: (+) ends at
cell periphery, (-) ends at centrosome

Question 8

what promotes nuceation at MTOC?

Answer 8

gamma-tubuliln
The PCM nucleates MTs via γ-tubulin and
several associated protein, aka the γ-tubulin
ring complex (γTuRC)
Anchors (-) ends to centrosome and overcomes
kinetic barrier to MT nucleation

Question 9

what must occur for bipolar mitotic spindle?

Answer 9

centrosome duplication

Question 10

iN VITRO RECONSTITUTION OF MOTILITY + DENTIFICATION OF THE MOTOR

Answer 10

Mixing charged latex beads with axoplasm,
microtubules, and ATP reconstitutes motility
Non-hydrolysable ATP locks beads in place

motor Strategy:
1. Produce lots of axoplasm
2. “Lock” motor onto MTs with non-
hydrolysable ATP analog (AMP-PNP)
3. Pellet MTs
4. Release motors by adding ATP
5. Fractionate (columns, etc) and test
fractions for motility in vitro

Question 11

THE MICROTUBULE-ASSOCIATED MOTOR

Answer 11

KINESIN

Question 12

Kinesin structure

Answer 12

Question 13

KINESIN BIOCHEMICAL CYCLE

Answer 13

ATP BINDING GENERATES FORCE

“Neck linker” zips onto head
domain in the ATP state
ATP hydrolysis decreases
affinity of head for neck linker
and MT
ADP-bound head is pulled
forward by zipping of the other
head

Question 14

MICROTUBULES IN NEURONS

Answer 14

(+) ends extend down axon
MTOC is the major site of nucleation
early in differentiation
Later on, most MTs are nucleated by
γ-tubulin at other sites or
transported from the cell body by
kinesin
Dendritic MTs have mixed polarity

Question 15

toward which end do kinesin motor proteins move?

Answer 15

positive

Question 16

Is there a minus-end directed protein motor?

Answer 16

yes, dynein
1.4 Megadaltons
Unrelated to kinesin
AAA domains bind ATP
Walks backwards!

Question 17

DYNACTIN

Answer 17

iS AN OBLIGATE COFACTOR OF DYNEIN

Dynactin: Complex of 23 polypeptides, 11 different proteins
Required for cargo binding and some motor functions

Question 18

COMPLEMENTARY ROLES IN AXONAL TRANSPORT

Answer 18

Kinesin: Delivery of vesicles, RNA, trophic
factors to synapse
Dynein: Retrieval of receptors,
membranes, other materials from synapse

Question 19

is microtuble polymerization a stable process?

Answer 19

no, governed by dynamic instability

Question 20

from where/what does dynamic instability of microtubules originate?

Answer 20

GTP hydrolysis
Rate of GTP hydrolysis increases upon
polymerization
GTP-tubulin is only found near the +-
end
GDP-tubulin is “bent” and under strain
“GTP-cap” holds the filament together
Losing the cap allows the filament to
depolymerize rapidly

Question 21

SELECTIVE BINDING TO THE GROWING OR STABILIZED PLUS END

Answer 21

Plus-end binding proteins [(+)-TIPs]
selectively bind the growing tip of
a microtubule.
EB1 is an essential (+)-TIP.
“Surfs” along tip of growing
microtubule.

Question 22

FUNCTIONS OF (+)-TIPS

Answer 22

EB1 recruits other proteins to the (+)
end: Dynactin, CLIP-170, CLASP,
APC.
Important for motor interactions, MT
stability, membrane binding, signal
transduction, and kinetochore
attachment

Question 23

What types of things does Actin allow cells to do?

Answer 23

Detect the signals
Change shape
Generate motile force
Reorient as needed
Phagocytose the bug

Question 24

DISCOVERY OF “MYOSIN”

Answer 24

Identified an abundant protein
component of muscle in high-salt
extracts.
Two forms, called myosin A (low
viscosity) and myosin B (high viscosity).
Squirting myosin B into low-salt buffer
creates “threads”.

Question 25

DISCOVERY OF “ACTOMYOSIN

Answer 25

A third, mystery protein from highly-purified tissue fractions converts myosin A into myosin B.
Called the mystery protein “actin” for its “activating” properties.
Actomyosin threads contract in the presence of ATP.
Created a model:
Myosin B is just myosin A bound to actin.
These “actomyosin” filaments are the structures observed in muscle.
Actomyosin filaments use ATP hydrolysis to drive muscle contraction.

Question 26

BETA-ACTIN

Answer 26

47 kD globular protein
4-7 nm diameter
ATP binding
100 mM in non-muscle cells
80% identity from yeast to human
Usually bound to profilin, a
nucleotide exchange factor

Question 27

F-ACTIN FILAMENTS

Answer 27

Filaments are polarized:
“Barbed” vs. “Pointed” ends
Helical structure: Repeats every
37 nm
~1/4 the diameter of a
microtubule
Far less rigid than microtubules

Question 28

Actin nucleation

Answer 28

Question 29

how do actin filaments grow/shrink?

Answer 29

Actin filaments tend to grow at the
barbed end, shrink at the pointed
end.

ATP HYDROLYSIS STARTS A TIMER FOR FILAMENT DISASSEMBLY

Question 30

• How do you overcome the kinetic barrier to nucleate a new filament?

Answer 30

ACTIN NUCLEATORS OVERCOME THE KINETIC BARRIER

All nucleators increase the likelihood of actin seed formation
Arp2/3 complex = most common
ARP2/3 NUCLEATES FILAMENTS AT BRANCHPOINTS
7 proteins, ~250 kD
Arp2 and 3 (“actin related protein”) mimic an actin dimer and reduce the
barrier to nucleation

Question 31

how do listeria avoid antibiotics?

Answer 31

LISTERIA “ROCKET” ON ACTIN TAILS

Question 32

ARP2/3 cofactors

Answer 32

ActA brings together Arp2/3
and actin monomers to nucleate
branched filaments.
Membrane localization of ActA
promotes filament assembly at
the bacterial cell surface.
Polymerization generates force!

Question 33

EUKARYOTIC NUCLEATION PROMOTING FACTORS

Answer 33

NPFs in the host cell and bacterial ActA
nucleate actin through similar mechanisms:
Pro/WH2 domains bind profilin and actin
monomers
CA domain binds Arp2/3
PIP2- and Cdc42-binding domains in
eukaryotic NPFs provide additional levels
of regulation

Question 34

NPFS LOCALIZE POLYMERIZATION TO THE MEMBRANE

Answer 34

Intra- or intermolecular domain
interactions inhibit WASP
Cdc42/PIP2 binding relieves
inhibition
Places actin regulation under
signal transduction control
Activation requires membrane
association!

polarized toward leading edge of moving cell

Question 35

RECONSTITUTION OF ROCKETING FROM PURIFIED COMPONENTS

Answer 35

ActA-coated beads mixed with:
Actin (fluorescently tagged)
Arp2/3
Cofilin
Profilin
Capping protein

Question 36

where is actin found in extending neuron?

Answer 36

@ growth cone

Question 37

MEMBRANE EXTENSIONS ARE GENERATED NEAR REGIONS OF RESTRICTED FLOW

Answer 37

F-actin accumulates
under bead
Trailing MTs extend to
anchor point
Small lamellae extend
outward from bead

Question 38

Myosin-neuron function

Answer 38

MYOSIN CONTRACTION HELPS STEER THE AXON