Transcript Microtubules, Motors and Membranes

Microtubules, Motors and
Membranes
Transport on Microtubules
• In neurons there is visible transport of vesicles
from cell body to growth cone
– Transcription and translation and membrane
biosynthesis in cell body
– Need to get material to growth cone to elongate
• Axonal transport
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Fast anterograde (3µm/sec)-vesicles
Intermediate anterograde (0.6µm/sec)-mitochondria
Slow anterograde- (0.002-0.03µm/sec) -proteins
Retrograde- 2µm/sec
Fast axonal transport movie
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Vesicles on microtubule’s in vitro
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Kinesin animation
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• Giant Squid axoplasm can be extruded and watched under
microscope
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Can watch vesicles move on mt’s
Now use brain mt’s and squid axoplasm
vesicles move with ATP added (2µm/sec)
vesicles bind but don’t move with AMPPNP
now isolate proteins that bind to mt’s in the presence of AMPPNP
but elute with ATP!
• Kinesin is discovered
Kinesins
• Moves toward the (+) end (1-2 µm/sec)
• 2 x 124kd + 64kd complex
• Double headed ATP motor with a tail that
binds cargo
• 4 families involved in vesicle movement
• 3 families involved in spindle function
• Some are actually (-) end directed
Heavy chains
Light chain
Flexible hinge
Head
Stalk
Tail
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Dynein
• Huge protein complex
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2-3 500 kd proteins
several intermediate and light chains
dynactin complex
4 proteins including an actin-related protein (ARP)
regulates dynein?
• (-) end directed ATPase motor (1-14µm/sec)
• Three classes of cytoplasmic plus flagellar
– One looks vesicular
– One is near Golgi
– One is in punctate structures of unknown origin
Microtubule motors in vitro
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1- Put motor on slide and add microtubules- the motor pushes the
microtubule along
2- add vesicles to microtubules on slide and the vesicles are moved on
the microtubules
Terasaki et al.
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DiOC6 stains mitochondria +ER
Shows a reticular network in cells
Co-localizes with mt’s
Depolymerize mt’s and it collapses, but
slower than mt’s
• During regrowth, the ER follows the mt’s
DiOC6 stain of NIH3T3 cell
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Dabora and Sheetz
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Make an membrane prep from CEF cells
Add to mt’s on coverslip
the vesicles are pulled out into a reticular network
requires ATP
inhibited by AMPPNP and vanadate (requires
kinesin and dynein motors)
• looks like Terasaki’s ER
• Recent- Kinesin binding protein found on
cytoplasmic face of ER- Kinectin
Turner and Tartakoff
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Depolymerize mt’s with nocodazole
Golgi vesiculates
required energy
Now remove nocodazole
mt’s reform
Golgi coalescence
requires energy
Golgi (green)
Microtubules’s (red)
Add nocodazole
ER-Golgi- Transport
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ER to Golgi traffic visualized in living cells John F. Presley, Nelson B. Cole,
Trina A. Schroer, Koret Hirschberg, Kristien Zaal and Jennifer LippincottSchwartzNature,Volume 389, Pages 81-85, 1997
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Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi
to Plasma Membrane Transport Intermediates in Living Cells J. Cell Biol.,
Volume 143, Number 6, 1998 1485-1503 Koret Hirschberg, Chad M. Miller,
Jan Ellenberg, John F. Presley, Eric D. Siggia, Robert D. Phair,§ and
Jennifer Lippincott-Schwartz
• Use GFP probes to visualize ER to Golgi transport
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VSVG ts mutant- doesn’t fold correctly at low temperature
40°C- blocked in ER
15°C- blocked in pre-Golgi
32°C- transports normally
Hirschberg et al.
Cells incubated at
40C overnight to
block in ER and then
released to visualize
dynamics of transport
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Presley et al. Cells held at 40 to block in ER
and then released to see movement to Golgi
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Presley et al. Cells blocked in pre-golgi at 15
then released to 32 to see movement to Golgi
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Presley et al. Movement of pre-golgi
structures (15C) in the presence of nocodazole
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Presley et al. Overexpress
Dynamitin at 15C and then shift
to 32 (release to Golgi)
Mitochondria and Microtubules
• Mitochondria also coalign with Mt’s in cell
• They are elongated into tubules along the
length of mT’s
• Recently, a kinesin homolog (Kif1B) has
been found to be specific for mitochondria
Mitochondria in Epithelial cell
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Lysosomes and Microtubules
• Label endosomal system see extensive
network of vesicles and tubules clustered
around MTOC
• Nocodazole causes dispersal
• Movements of individual vesicles ceases
when mt’s depolymerized
• repolymerize mt’s and they recluster at
MTOC
Melanophores
• Vesicles move bidirectionally on Mt’s to change
the color of cells in fish scales
– Melatonin stimulating hormone causes vesicles to
disperse
• cAMP increases
– Melatonin causes them to aggregate near the nucleus
• cAMP decreases
• Dynein does inward movement
• Kinesin + Myosin V does outward
Models for
bidirectional vesicle
transport (Gross et al. The
Journal of Cell Biology,
Volume 156, Number 4,
February 28, 2002 715–724)
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Melanophore papers
• Movements of melanophores in cells
• Functional Coordination of
Microtubule and Actin Based Motility
in Melanophores. V. I. Rodionov, A. J.
Hope, T. M. Svitkina and G.G. Borisy
Curr. Biol., 8(3): 165-168, 1998
Melanophore aggregation
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Melanophore dispersion
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Movements are
microtubule dependent
Gross et al. The Journal of Cell
Biology, Volume 156, Number 5,
March 4, 2002 855–865
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Melanophore dispersion after
Lantrunculin treatment
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All three motors stay bound to melanosomes during movement
(Gross et al.
The Journal of Cell Biology, Volume 156, Number 5, March 4, 2002 855–865
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Dynactin complex
(Deacon et al. The Journal of Cell Biology,
Volume 160, Number 3, February 3, 2003 297–301)
• Complex that binds dynein to cargo
• Inhibition of function by overexpressing
dynamitin ( a component of dynactin complex)
inhibits both aggregation and dispersal
• Found dynamitin binds to Kinesin as well as
dynein
• Thus the dynactin complex may regulate both with
some kind of bidirectional on/off switch
Vesicle trafficking: Golgi-TGNsorting in epithelial cells
• Problem: All secretory, lysosomal,
membrane proteins sort from ER to Golgi
and then are distributed in the TGN to
different sites
• How is the sorting accomplished?
• In Epithelial cells you sort cargo to the
apical and basolateral membranes. How?
Use fluorescent labeled markers that sort to apical (CFP) or
basolateral (YFP) part of the cell. Keller et al. Nature Cell
Biology, 3:140-147 2001
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Transfect in probes
Block transport at low temperature
Release block and image
Most probe is initially in the same vesicle (yellow)
Over time, they sort into red and green vesicles
Can see tubules pull out of a single sorting
compartment that are either red or green
• Mechanism unknown
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Movements near the membrane are not
diffusion! (Keller et al.)
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TIRF imaging of
basolateral side of cell
Red- endocytic marker
(dextran uptake)
Green- basolateral
secretory marker
Membrane Tension
• Membranes seem to be under tension-note the
pulling out of membranes along microtubule’s in
vitro looks like ER
• Sorting seems to involve stretching of the sorting
compartment membranes into tubules
• There is now evidence that in the TGN, you don’t
bud off small vesicles, but large interconnected
networks of tubules and vesicles
• Brefeldin A Blinkout
– Brefeldin A blocks the transport from ER to Golgi
– Does not block recycling from Golgi to ER
– Add bfa and watch the Golgi disperse- it is not gradual,
it is explosive!!
Golgi Blinkout with Brefeldin A
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Future?
• Challenge is to figure out the specific
function of each motor
– Where is it?
– What is it’s cargo?
– What turns the motor off and on?
• Organization of Golgi, ER, lysosomes by
motors and how function is interrelated