Division of Intramural Research
Laboratory of Molecular Cardiology
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Cellular and Molecular Motility Section |
James R. Sellers,Ph.D.
Chief, Cellular and Molecular Motility Section
Phone: (301) 496-6887 or -5639
Fax: (301) 402-1542
Email: jsellers@helix.nih.go
Research Interests
Our research focuses on the structure, function
and regulation of myosins, a class of actin-dependent
motor proteins. There are currently at least eighteen classes of
myosin that have been identified by phylogenetic analysis of the motor
domains. In addition, the tails of each class of myosin have distinct
features that may include many possible functional motifs, such as SH3
domains, FERM domains, GAP domains, pleckstrin-homology domains and coiled-coiled
forming regions. The phylogenetic distribution of the various myosins
shows that no myosin class is universally present. Analysis of
the complete genome of yeast shows that there are five myosin genes: two
myosin I genes, one myosin II gene and two myosin V genes. Humans
have 39 myosin genes from 11 different classes. Human diseases
have been associated with mutations in several myosin genes, including
the beta-cardiac myosin II heavy chain and light chains (hypertrophic
cardiomyopathy), nonmuscle myosin IIA heavy chain (giant platelet disorders
and deafness) and the myosin VA heavy chain (Griscelli's syndrome). Mutations
in several other myosin heavy chain genes are also associated with deafness
including myosin IIIa (DFNB30), myosin VI (DFNA22), myosin
VIIa (Usher's IB syndrome), and myosin XV (DFNB3, deafness). Many
of the myosin classes are represented by multiple genes within a given
organism. We are very interested in the specific roles and functions
of this variety of myosins. We are studying myosins from class
I, II, III, V, IX, X and XV. The techniques that are used range
from biophysical studies of single isolated myosin molecules to genetic
analysis of myosin function in Drosophila. We routinely utilize the actin sliding in vitro motility
assay and steady-state enzymatic analysis to characterize myosins. We
are making extensive use of baculoviral expression of myosin in Sf9 cells
in order to prepare naturally occurring isoforms of myosin as well as
mutant myosins. For these studies, we use the available three-dimensional
structures of various myosin head fragments as a guide.
We have several concurrent
projects. One is to use baculoviral expression
of mutant myosins in order to study the molecular mechanisms
of regulation and of the effect of disease causing
mutations in nonmuscle myosin II isoforms. We
are also studying myosin III from Limulus in
collaboration with Barbara Battelle (Whitney Laboratory). This
myosin is found in the retinal cells of this organism.
We are very interested in how myosin V, a vesicle
or organelle motor, is able to move processively
on actin. To study this myosin, we are using
several state-of-the-art biophysical techniques
with Claudia Veigel and Justin Molloy (University
of York, UK). We are studying the mechanics
of the interaction of single myosin molecules
with actin using an optical trap. These
data show that myosin V can move processively
on actin taking 36 nm steps, but that this step
is a combination of a 25 nm powerstroke compared
with a thermal motion. Takeshi Sakamoto
in our lab has built a Total Internal Reflection
Fluorescences (TIRF) microscope which enables
us to see single GFP-labeled myosin molecules
moving processively on actin filaments. In
addition, we are collaborating with John Trinick,
Peter Knight and Stan Burgess (University of Leeds,
UK) to examine the conformation of myosin V bound
to actin while moving processively and of myosin
V alone. These images have allowed us to
see the myosin powerstroke for the first time.
We have two projects that involve studying myosin
mutations that result in human disease. One
of these studies mutations in the nonmuscle myosin
II heavy chain genes that are found in patients
with one of several giant platelet disorders including
May-Hegglin Anomaly, Sebastians Syndrome or Fechtner
Syndrome. In addition, some NMIIA mutations
are associated with familial deafness. The
second project is in collaboration with Tom Friedman
(NIDCD) and involves studies of the myosin XV
heavy chain gene. Mutations in this gene
give rise to hereditary deafness.
We have cloned the myosin V gene from Drosophila
and have a mutant fly line where the myoV gene
is effectively knocked out. We found that
myoV is abundantly present in the eyes of flies
and are attempting to determine its function. In
addition, we have cloned myosin VIIb from flies
and conducted an analysis of the myosin heavy
chain genes in the completed Drosophila genome.
Selected Recent
Publications
-
Wu,
X., Wang, F., Rao, K., Sellers, J.R. and Hammer, J.A. III: Rab27a is
an essential component of the melanosome receptor for myosin Va. Mol. Biol.
Cell, 13:1735-1749, 2002.
-
Boger,
E.T., Sellers, J.R. and Friedman, T.B.: Human myosin XVBP is a
transcribed pseudogene. J. Muscle Res. Cell Motil., 22: 477-483,
2002.
-
Wu,
X.S., Rao, K., Zhang, H., Wang, F., Sellers, J.R., Copeland, N.G., Jenkins,
N.A. and Hammer, J.A. III: Identification of an organelle receptor
for myosin Va. Nature Cell Biol. 4, 271-278, 2002.
-
Veigel,
C., Wang, F., Bartoo, M.L., Sellers, J.R. and Molloy, J.E.: The gated
gait of the processive molecular motor, myosin V. Nature Cell
Biol. 4, 59-65, 2002.
-
Cremo,
C.R., Wang, F., Facemyer, K. and Sellers, J.R.:
Phosphorylation-dependent regulation is absent in a nonmuscle heavy meromyosin
construct with one complete head and one head lacking the motor domain.
J. Biol. Chem. 276, 41465-41472, 2001.
-
Yamashita,
R.A., Sellers, J.R. and Anderson, J.B.: Identification and analysis
of the myosoin superfamily in Drosophila: A database approach. J.
Muscle Res. Cell Motil. 21, 491-505, 2000.
-
Walker,
M.L., Burgess, S.A., Sellers, J.R., Wang, F., Hammer, J.A., III, Trinick, J.
and Knight, P.J.: Two-headed binding of a processive myosin to
F-actin. Nature 405, 804-807, 2000.
-
Ellison,
P.A., Sellers, J.R. and Cremo, C.R.: Kinetic properties of smooth
muscle heavy meromyosin with one-head thiophosphorylated. J. Biol. Chem.
275, 15142-15151, 2000.
-
Wang,
F., Harvey, E.V., Conti, M.A., Wei, D. and Sellers, J.R.: A conserved
negatively charged amino acid modulates function in human nonmuscle myosin
IIA. Biochemistry 39, 5555-5560, 2000.
-
Sellers,
J.R.: Myosins: a diverse superfamily. Biochim.
Biophys. Acta. 1496, 3-22, 2000.
-
Wang,
F., Chen, L., Arcucci, O., Harvey, E.V., Bowers, B., Xu, Y., Hammer, J.A., III
and Sellers, J.R.: Effect of ADP and ionic strength on the kinetic
and motile properties of recombinant mouse myosin V. J.Biol. Chem.
275, 4329-4335, 2000.
-
Kudryashov,
D.S., Chibalina, M.V., Birukov, K.G., Lukas, T.J., Sellers, J.R., Van Eldik,
L.J., Watterson, D.M. and Shirinsky, V.P.: Unique sequence of a high
molecular weight myosin light chain kinase is involved in interaction with
actin cytoskeleton. FEBS Lett. 463, 67-71, 1999.
-
Friedman,
T.B., Sellers, J.R. and Avraham, K.B.: Unconventional myosins and the
genetics of hearing loss. Am. J. Hum. Genet. 89, 147-157,
1999
-
Liang,
Y., Wang, A., Belyantseva, I.A., Anderson, D.W., Probst, F.J., Barber, T.D.,
Miller, W., Touchman, J.W., Jin, L., Sullivan, S.L., Sellers, J.R., Camper,
S.A., Lloyd, R.V., Bachar, B., Friedman, T.B. and Fridell, R.A.:
Characterization of the human and mouse unconventional myosin XV genes
responsible for hereditary deafness DFNB3 and Shaker 2. Genomics
61, 243-258, 1999.
- Sellers, J.R.: Unphosphorylated crossbridges
and latch: smooth muscle regulation revisited. J. Muscle Res.
Cell Motil. 20, 347-349, 1999.
- Sellers, J.R.: Protein Profile:
Myosins. 2nd Ed. Oxford Univ. Press, Oxford, 1999.
-
Wang,
Z.-Y., Wang, F., Sellers, J.R., Korn, E.D. and Hammer, J.A., III:
Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by
using site-directed mutagenesis. Proc. Natl. Acad. Sci. (U.S.A.)
95, 15200-15205, 1998.
- Bonafe, N.M. and Sellers, J.R.:
Calmodulin-binding proteins of the cytoskeleton. In Calmodulin and Signal
Tranduction, L. Van Eldik and D.M. Watterson, Editors. Academic Press,
San Diego. 347-395, 1998.
-
Jenkins,
A.L., Nannizzi-Alaimo, L., Silver, D., Sellers, J.R., Ginsberg, M.H., Law, D.A.
and Phillips, D.R.: Tyrosine phosphorylation of the beta3 cytoplasmic
domain mediates integrin-cytoskeletal interactions. J. Biol. Chem.
273, 13878-13885, 1998.
- Sellers, J.R. and Jiang, H.: In vitro
motility assays with actin. in Cell Biology: A Laboratory Handbook,
2nd Ed., J.Celis, Editor. Academic Press, San Diego. Vol. 2,
336-343, 1998.
-
Sweeney,
H.L., Rosenfeld, S.S., Brown, F., Faust, L., Smith, J., Xing, J., Stein, L.A.
and Sellers, J.R.: Kinetic tuning of myosin via a flexible loop
adjacent to the nucleotide binding pocket. J. Biol. Chem. 273,
6262-6270, 1998.
-
Han,
Y.J. and Sellers, J.R.: Motility assays on molluscan native thick
filaments. Methods Enzymol. 298, 427-435, 1998.
-
Battelle,
B.A., Andrews, A.W., Calman, B.G., Sellers, J.R., Greenberg RM, Smith
WC: A myosin III from Limulus eyes is a clock-regulated
phosphoprotein. J. Neurosci. 18, 4548-4559, 1998.
-
Bonafe,
N. and Sellers, J.R.: Molecular characterization of myosin V from
D. melanogaster. J. Muscle Res. Cell Motil. 273,
6262-6270, 1998.
-
Shah,
A.M., Mebazaa, A., Yang, Z.K., Cuda, G., Lankford, E.B., Pepper, C.B., Sollott,
S.J., Sellers, J.R., Robotham, J.L. and Lakkatta, E.G.: Inhibition of
myocardial crossbridge cycling by hypoxic endothelial cells: a potential
mechanism for matching oxygen supply and demand? Cir. Res.
80, 688-698, 1997.
-
Ford,
H.L., Silver, D.L., Kachar, B., Sellers, J.R. and Zain, S.B.: Effect
of Mts1 on the structure and activity of nonmuscle myosin II.
Biochemistry36, 16321-16327, 1997.
-
Silver,
D.L., Vorotnikov, A.V., Watterson, D.M., Shirinsky, V.P. and Sellers,
J.R.: Sites of interaction between kinase-related protein and smooth
muscle myosin. J. Biol. Chem. 272, 25353-25359, 1997.
-
Pohl,
J., Winder, S.J., Walsh, M.P., Sellers, J.R. and Gerthoffer, W.T.:
Phosphorylation of calponin in airway smooth muscle. J. Physiol.
(London) 272, 113-123, 1997.
-
Wang,
Z.-Y., Sakai, J., Matsudaira, P.T., Baines, I.C., Sellers, J.R., Hammer, J.A.
III and Korn, E.D.: The amino acid sequence of the light chain of
Acanthamoeba myosin IC. J. Muscle Res. Cell Motil. 18,
395-398, 1997.
-
Cuda,
G., Pate, E., Cooke, R. and Sellers, J.R.: In vitro actin filament
sliding velocities produced by mixtures of different types of myosin.
Biophys. J. 72, 1767-1779, 1997.
-
Cuda,
G., Fananapazir, L., Epstein, N.D. and Sellers, J.R.: The in
vitro motility activity of ß-cardiac myosin depends on the nature of
the ß-myosin heavy chain gene mutation in hypertrophic cardiomyopathy.
J. Muscle Res. Cell Motil. 18, 275-283, 1997.
-
Gerthoffer,
W.T., Yamboliev, I.A. Shearer, M., Pohl, J., Haynes, R., Dang, S., Sato, K. and
Sellers, J.R.: J. Physiol. (Lond.) 495, 597-609,
1996.
-
Kelley,
C.A., Sellers, J.R., Gard, D.L., Bui, D., Adelstein, R.S. and Baines, I.C.:
Xenopus nonmuscle myosin heavy chain isoforms have different subcellular
localizations and enzymatic activities. J. Cell Biol. 134,
675-687, 1996.
-
Olney,
J., Sellers, J.R. and Cremo, C.R.: Structure and function of the 10S
conformation of smooth muscle myosin. J. Biol. Chem.271,
20375-20384, 1996.
-
Sellers,
J.R.: NCD and kinesin: Two structural cousins of myosin. J. Muscle
Res. Cell Motil. 17, 173-176, 1996.
-
Poetter,
K., Jiang, H., Hassanzadeh, S., Master, S.R., Chang, A., Dalakas, M.C.,
Rayment, I., Sellers, J.R., Fananapazir, L. and Epstein, N.D.:
Mutations in either the essential or regulatory light chains of myosin are
associated with a rare myopathy in human heart and skeletal muscle. Nature
Genet. 13, 63-69, 1996.
-
Sellers,
J.R., Goodson, H.V. and Wang, F.: A myosin family reunion. J.
Muscle Res. Cell Motil. 17, 7-22, 1996.
-
Pato,
M.D., Sellers, J.R., Preston, Y.A., Harvey, E.V. and Adelstein, R.S.:
Baculovirus expression of chicken nonmuscle heavy meromyosin II-B:
Characterization of alternatively spliced isoforms. J. Biol. Chem.
271, 2689-2695, 1996.
Group Members
Estelle V. Harvey
John Limouze
Ella Peresechensky
Takeshi Sakamoto, Ph.D.
Fei Wang, Ph.D.
Yue Zhang
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