Douglass Flashcards
OPTICAL BIREFRINGENCE
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
SHINYA INOUE AND THE DISCOVERY OF MICROTUBULES
Two assumptions:
(1) Cellular birefringent structures
might be important.
(2) We might learn something by
watching them change over time.
saw mitotic spindles
______ DISRUPTS THE MITOTIC SPINDLE
colchicine
COLCHICINE AND THE IDENTIFICATION OF TUBULIN
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.
Tubulin dimers
heterodimers
Two isoforms of tubulin: αand β
50% sequence identity
Typically exist as a heterodimer:
Kd = 84 nM
Bind to and hydrolyze GTP
Microtubule structure
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
CENTROSOME
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
what promotes nuceation at MTOC?
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
what must occur for bipolar mitotic spindle?
centrosome duplication
iN VITRO RECONSTITUTION OF MOTILITY + DENTIFICATION OF THE MOTOR
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
THE MICROTUBULE-ASSOCIATED MOTOR
KINESIN
Kinesin structure
KINESIN BIOCHEMICAL CYCLE
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
MICROTUBULES IN NEURONS
(+) 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
toward which end do kinesin motor proteins move?
positive
Is there a minus-end directed protein motor?
yes, dynein
1.4 Megadaltons
Unrelated to kinesin
AAA domains bind ATP
Walks backwards!
DYNACTIN
iS AN OBLIGATE COFACTOR OF DYNEIN
Dynactin: Complex of 23 polypeptides, 11 different proteins
Required for cargo binding and some motor functions
COMPLEMENTARY ROLES IN AXONAL TRANSPORT
Kinesin: Delivery of vesicles, RNA, trophic
factors to synapse
Dynein: Retrieval of receptors,
membranes, other materials from synapse
is microtuble polymerization a stable process?
no, governed by dynamic instability
from where/what does dynamic instability of microtubules originate?
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
SELECTIVE BINDING TO THE GROWING OR STABILIZED PLUS END
Plus-end binding proteins [(+)-TIPs]
selectively bind the growing tip of
a microtubule.
EB1 is an essential (+)-TIP.
“Surfs” along tip of growing
microtubule.
FUNCTIONS OF (+)-TIPS
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
What types of things does Actin allow cells to do?
Detect the signals
Change shape
Generate motile force
Reorient as needed
Phagocytose the bug
DISCOVERY OF “MYOSIN”
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”.
DISCOVERY OF “ACTOMYOSIN
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.
BETA-ACTIN
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
F-ACTIN FILAMENTS
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
Actin nucleation
how do actin filaments grow/shrink?
Actin filaments tend to grow at the
barbed end, shrink at the pointed
end.
ATP HYDROLYSIS STARTS A TIMER FOR FILAMENT DISASSEMBLY
- How do you overcome the kinetic barrier to nucleate a new filament?
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
how do listeria avoid antibiotics?
LISTERIA “ROCKET” ON ACTIN TAILS
ARP2/3 cofactors
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!
EUKARYOTIC NUCLEATION PROMOTING FACTORS
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
NPFS LOCALIZE POLYMERIZATION TO THE MEMBRANE
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
RECONSTITUTION OF ROCKETING FROM PURIFIED COMPONENTS
ActA-coated beads mixed with:
Actin (fluorescently tagged)
Arp2/3
Cofilin
Profilin
Capping protein
where is actin found in extending neuron?
@ growth cone
MEMBRANE EXTENSIONS ARE GENERATED NEAR REGIONS OF RESTRICTED FLOW
F-actin accumulates
under bead
Trailing MTs extend to
anchor point
Small lamellae extend
outward from bead
Myosin-neuron function
MYOSIN CONTRACTION HELPS STEER THE AXON
