Dr. Richard B. Vallee, Ph. D.
rv2025@columbia.edu
Microtubule Motors, Brain Developmental Disease, Endocytosis
Cytoplasmic dynein.
Microtubules are involved in mitosis, neuronal differentiation, axoplasmic
transport, secretion, endocytosis, and many other basic cellular processes. How
microtubules perform their varied functions is a major interest of our
laboratory. We have identified and purified a large motor protein, cytoplasmic
dynein, which produces force toward the minus end of microtubules. Existing
evidence indicates that cytoplasmic dynein is responsible for retrograde axonal
transport as well as the positioning and organization of the Golgi apparatus,
endosomes, lysosomes, and other membranous organelles. Cytoplasmic dynein also
functions during mitosis in spindle assembly and orientation, and in the
capture and movement of chromosomes (see figure 1). It has
been implicated in the reorganization of microtubules during wound healing and
in the migration of neurons during brain development. Recent evidence
indicates that it is also used by a number of viruses to travel to the
nucleus.
Our laboratory is interested in how cytoplasmic dynein is targeted to the
large diversity of subcellular structures to which attaches, and how it
produces force. Cytoplasmic dynein is a 1.2 mDa complex containing two heavy
chains (HCs, 532 kDa). Each of these subunits contains a motor domain
responsible for ATP hydrolysis and force production. A variety of accessory
subunits, the intermediate, light intermediate, and light chains function in
cargo binding, along with another large complex, dynactin. Our lab has
developed tools to interfere with dynein and dynactin activity to explore their
functions in neuronal and nonneuronal cells. We are also interested in
identifying the "receptors" for dynein and dynactin associated with diverse
forms of cargo, and in learning how dynein targeting is regulated.
Little is known about the mechanism of dynein force production. The motor
domain is ten-fold larger than that of kinesin, but shares no apparent
structural similarity. Current evidence indicates that the dynein motor domain
consists of a series of six AAA ATPase units possibly arranged in a circle.
Protruding from this structure is an unusual 10-12 nm stalk, at the tip of
which lies the microtubule binding site. Our current evidence suggests that
the stalk consists of an antiparallel coiled-coil α-helix. We are interested
in defining the structural organization of this and the other components of the
dynein motor domain more precisely, in learning the role of the multiple ATPase
modules, and in determining how conformational information from is transmitted
from them through the stalk to the microtubule binding site.
Role of cytoplasmic dynein in the brain developmental disease lissencephaly.
Neuronal migration represents a critical step in brain development. It
occurs after neuronal/glial progenitor cells have completed their final
division within the ventricular and subventricular zones (Fig.
4A). Neurons then migrate outward to establish the characteristic cortical
layers, but how this process is regulated is poorly understood. Lissencephaly
is a developmental disease involving abnormal neuronal migration. Children
born with this condition lack the convolutions normally present on the surface
of the brain, and the distribution of neurons within the cerebral cortex is
randomized. The Miller-Dieker form of lissencephaly has been shown to result
from null mutations in the LIS-1 gene. Lower eukaryotic homologues of LIS-1
have been implicated in cytoplasmic dynein function, but it has been uncertain
whether this is also true for vertebrate LIS1 which has other known
functions.
We find that LIS-1 interacts with both cytoplasmic dynein and dynactin and
colocalizes with these proteins at mitotic kinetochores, at the cortex of
mitotic cells, and at the tips of growing microtubules. Consistent with a
mitotic function, overexpression of full-length or truncation mutant forms of
LIS-1 in cultured mammalian cells dramatically inhibits mitosis (mitotic index
>30%). Spindle defects include unatttached chromosomes, multiple spindle
poles, and hyperelongated astral microtubules. Cytoplasmic dynein and dynactin
staining in the cell cortex is disrupted, and mitotic spindle orientation is
randomized (Fig. 4B, C). Anti-LIS-1 antibody injection
also interferes with mitosis, specifically with chromosome alignment and
segregation. LIS1 overexpression affects only a subset of dynein functions in
our hands, as Golgi and endosome organization appear to be normal. We speculate
that mutations in the LIS-1 gene may affect brain development through effects
on neuronal progenitor cell division as well as other aspects of progenitor
cell motility. Our goals are to define the molecular mechanisms by which LIS-1
interacts with and regulates cytoplasmic dynein and the detailed mechanism by
which LIS1 regulates cell division and neuronal migration.
The role of dynamin in endocytosis and synaptic vesicle recycling.
Dynamin is an ~100 kDa GTPase identified in this lab, which is involved in the
initial stages of endocytosis (see figures 2 and 3).
Dynamin assembles to form a helical polymer at the cell surface surrounding the
neck of coated and non-coated pits. It is uncertain whether the polymer serves
a mechanical or a regulatory role in membrane budding. Mutations in the GTPase
domain of dynamin dominantly inhibit endocytosis and demonstrate that dynamin
serves as a master control factor for regulating the entry of macromolecules
and viruses into the cell. Temperature-sensitive mutations in Drosophila
dynamin (shibire) produce rapid, reversible paralysis due to a failure
in synaptic vesicles to reform from the plasma membrane once they have
discharged their contents. Dynamin consists of an N-terminal GTPase domain,
and contains a pleckstrin homology (PH) domain, and a 100 a.a. C-terminal
basic, proline-rich domain. We have produced an extensive range of dynamin
mutant constructs for analysis of the role of the protein in endocytosis,
membrane binding, self-association, and interaction with other proteins. We
have found that a C-terminal 66 a.a. region is required for clathrin
co-localization and for SH3 domain binding, suggesting that SH3-containing
proteins link dynamin to coated pits. We have identified three regions of the
dynamin polypeptide that each form homotetrameric α-helical coiled-coils,
suggesting that the dynamin tetramer is organized around a parallel coiled-coil
shaft. We have recently found an interaction between the dynamin-2 gene
product and members of the Shank family, scaffolding proteins associated with
the post-synaptic density and cortical actin cytoskeleton. The
Shank-associated dynamin pool may be responsible for glutamate receptor
down-regulation and other aspects of postsynaptic membrane remodeling. We
hope to test this possibility and the role of coiled-coil switching in dynamin
assembly and membrane scission.
Figures
Figure One Legend:
Effect of dynactin disruption on dividing COS-7 cells. Cell at left is
overexpressing the dynamitin subunit of the dynactin complex; cell at right is
a non-expressing control. Blue, anti-dynamitin; green, anti-tubulin; red, CREST
auto-immune anti-centromere. Dynactin overexpression inhibits mitosis during
prometaphase and distorts the mitotic spindle (from Echeverri et al.,
1996).
Figure Two Legend:
Diagram of dynamin structural domains:
GTPase, GTPase domain with consenses sequence elements indicated;
CC, α-helical coiled-coil regions
as shown by circular dichroism; PH,
pleckstrin homology domain; PRD, basic, proline-rich domain with
SH3-binding polyproline helices indicated. Amino acid number for rat dynamin 1b
is shown at bottom. (see Okamoto et al., 1999)
Figure Three Legend:
Negative stain electron micrograph of polymer (outer diameter ~ 50 nm) assembled in
vitro from rat dynamin-1b expressed using baculovirus (courtesy of C. Gamby
and G. Hendricks).
Figure Four Legend
A) Neuronal progenitor cells at progressive stages of development in the
vertebrate ventricular zone. Aspects of cell motility potentially affected by
LIS-1 mutations are shown. Apical (ventricular) surface at bottom. B and C)
Dividing polarized MDCK epithelial cells examined by confocal anti-tubulin
immunofluorescence microscopy. B) Control mitotic spindle lies parallel to
coverslip, as shown. C) Mitotic spindle in LIS-1 overexpressing cell is
misoriented. Regulation of spindle orientation by LIS1 is proposed to control
affect the timing of neurogenesis in the developing brain and the subsequent
destination of migrating neurons. (from Faulkner and Vallee, 2000).
Selected Publications
Holzbaur, E. L. F., Hammarback, J. A., Paschal, B. M.,
Kravit, N. G., Pfister, K. K., and Vallee, R. B. (1991) Homology of a 150 kD
Cytoplasmic Dynein-associated Polypeptide with the Drosophila Gene Glued.
Nature, 351: 579-583.
Chen, M. S., Obar, R. A., Schroeder, C. C., Austin, T. W.,
Poodry, C. A., Wadsworth, S. C., and Vallee, R. B. (1991) Multiple Forms of
Dynamin are Encoded by shibire, a Drosophila Gene Involved in Endocytosis.
Nature, 351: 583-586.
Shpetner, H. S., and Vallee, R. B. (1992) Dynamin is a
GTPase which is Stimulated to High Levels of Activity by Microtubules.
Nature, 355: 733-735.
Vallee, R. B., and Sheetz, M. P. (1996) Targeting of Motor
Proteins. Science, 271: 1539-1544.
Gee, M.A., Heuser, J. E., and Vallee, R.B. (1997) An
Extended Microtubule-binding Structure within the Dynein Motor Domain
Nature, 390: 636-639.
Vallee, R. B., and Gee, M. A. (1998) Make Room for Dynein. Trends
Cell Biol., 8: 490-494.
Okamoto, P. M., Tripet, B., Litowski, J., Hodges, R. S., and
Vallee, R. B. (1999) Multiple Distinct Coiled-coils Mediate Dynamin
Self-assembly. J. Biol. Chem., 274: 10277-10286.
Purohit, A., Tynan, S. H., Vallee, R. B., and Doxsey, S.
(1999) Direct Interaction of Pericentrin with Cytoplasmic Dynein Light
Intermediate Chain Contributes to Mitotic Spindle Organization. J. Cell
Biol.,147: 481-492.
Garces, J. A., Clark, I. B., Meyer, D. I., and Vallee, R. B.
(1999) Interaction of the p62 Subunit of Dynactin with Arp1 and the Cortical
Actin Cytoskeleton. Curr. Biol., 9: 1497-1500.
Ye, G.-J., Vaughan, K. T., Vallee, R. B., and Roizman, B.
(2000) The Herpes Simplex Virus 1 UL34 Protein Interacts with a
Cytoplasmic Dynein Intermediate Chain and Targets Nuclear Membrane. J.
Virol., 74: 1355-1363.
Kreitzer, G., Marmorstein, A., Okamoto, P. M., Vallee, R.
B., and Rodriguez-Boulan, E. (2000) Kinesin and Dynamin Are Required for
Post-Golgi Transport of a Plasma Membrane Protein. Nature Cell
Biol., 2: 125-127.
Tynan, S. H., Gee, M. A., and Vallee, R. B. (2000) Distinct
but Overlapping Sites for Intermediate Chain Binding, Light Intermediate Chain
Binding, and Dimerization within the Cytoplasmic Dynein Heavy Chain. J.
Biol. Chem., 275: 32769-74.
Faulkner, N. E., Dujardin, D. L., Tai, C.-Y., Vaughan, K.
T., O’Connell, C. B., Wang, Y.-L., and Vallee, R. B. (2000) A role for
the lissencephaly gene LIS-1 in mitosis and cytoplasmic dynein function. Nature
Cell Biol., 2: 784-791.
Vallee, R. B., Tai, C.-Y., and Faulkner, N. E. (2001) LIS1:
Cellular Function of a Disease-causing Gene. Trends Cell Biol., 11:155-160.
Joseph, H. J., Chen, Y.-J., Palazzo, A. F., Dujardin, D. L.,
Alberts, A. S., Pfister, K. K., Vallee, R. B., and Gundersen, G. G. (2001)
CDC42, Dynein and Dynactin Regulate MTOC Reorientation Independent of
Microtubule Stabilization. Curr. Biol., 11: 1536-1541.
Tai, C.-Y., Dujardin, D. L., Faulkner, N. E., and Vallee, R. B. (2002)
Role of Dynein, Dynactin, and CLIP170 Interactions in LIS1 Kinetochore
Function. J. Cell Biol., 156: 959-968.
Dujardin D. L., Barnhart L. E., Stehman S. A., Gomes E. R., Gundersen G. G., Vallee
R. B. (2003) A role for cytoplasmic dynein and LIS1 in directed cell
movement. J Cell Biol., 163(6): 1205-11.
Academic Background
Richard Vallee received his B. A. from Swarthmore College, and his Ph.
D. from Yale University.