Division of Intramural
Research
Laboratory of Cell Biology
Dr. Edward D. Korn, Chief
E-mail: edk@nih.gov
50 South Drive MSC 8017, Building 50, Room 2517, Bethesda,
MD 20892-8017
Phone: (301) 496-1616, FAX: (301) 402-1519
This Laboratory comprises four sections with six independent
investigators with research interests in a range of biophysical,
biochemical and cell biological problems including: bioenergetics;
heat shock proteins; membrane and organelle trafficking; cytoskeletal
rearrangements; regulation and function of actin-based motors
(myosins). All members of the Laboratory partake in several
weekly journal clubs, research meetings and seminars by invited
speakers and monthly meetings with the members of the NHLBI
Laboratory of Molecular Cardiology. In these ways, an intense
laboratory experience is complemented by continual exposure
to a spectrum of biochemical, biophysical, cell biological
and genetic science. Positions are always available for students
- high school, undergraduates and graduate - to work part
time or full time and for postdoctoral fellows from the United
States and other countries. Interested individuals should
contact one of the principal investigators whose research
interests are summarized below.
Staff
- Cellular
Biochemistry Section, Edward D. Korn, Ph.D. and Julie
G. Donaldson, Ph.D.
- Cellular Physiology Section,
Evan Eisenberg, M.D., Ph.D. and Lois Greene, Ph.D.
- Molecular Cell Biology Section,
John A. Hammer, III, Ph.D.
- Membrane Enzymology Section, Richard
W. Hendler, Ph.D.
Cellular
Biochemistry Section: Edward D. Korn, Ph.D. and Julie G. Donaldson,
Ph.D.
Edward D. Korn: Members of the very large
myosin superfamily have essential roles in multiple, critical
cellular processes of all eukaryotic cells including, but
not limited to, muscle contraction, cell motility, cytokinesis,
phagocytosis, organization of the cytoskeleton and movement
of intracellular organelles. Class-I myosins contain a single
heavy chain and one or more light chains while all other
myosins contain two identical heavy chains and at least
four light chains. Myosins are actin-based motors with actin-activated
ATPase activity that is coupled to mechanical and structural
events involving the actin filaments. The actin-dependent
ATPase activity of different myosins in nonmuscle cells
is regulated by phosphorylation of either the heavy chain,
the light chain, or both. We are particularly interested
in the mechanisms by which light and heavy chain phosphorylation
regulate the activity of Class-I and Class-II myosins through
cooperative interactions between the head and tail domains
of the heavy chains of these myosins. The kinase that phosphorylates
the heavy chain of ameboid class-I myosins is a member of
the PAK family - kinases that are activated by autophosphorylation
stimulated by binding of the small GTPases, Rac and Cdc42.
We are investigating the mechanism of this regulation and
the role of Paks in the reorganization of the cytoskeleton
of mammalian cells in culture. By extensive use of mutated
myosins and chimeras of head and tail regions of different
Class-I and Class-II myosins, we are correlating the biochemical
and biophysical properties of the pure proteins in vitro
to their ability to support specific cellular activities.
Currently, for example, we are investigating the requirements
for a Class-II myosin to support cytokinesis and differentiation
of Dictyostelium discoideum and, in collaboration with Dr.
Gregory May, University of Texas, the minimal requirements
for the essential function(s) of the single Class-I myosin
in Aspergillus nidulans.
Selected recent publications:
Brzeska, H. and Korn, E.D. (1996). Regulation of class-I
and class-II myosins by heavy chain phosphorylation. J.
Biol. Chem. 271:16983-16986. [PubMed]
Brzeska, H., Szczepanowska, J., Hoey, J., and Korn, E.D.
(1996). The catalytic domain of Acanthamoeba
myosin I heavy chain kinase. II. Expression of active
catalytic domain and sequence homology to p21-activated
kinase (PAK). J. Biol. Chem. 271:27056-27062.
[PubMed]
Zolkiewski, M., Redowicz, M. J., Korn, E. D., Hammer,
J. A., III and Ginsburg, A. (1997). Two-state unfolding
of a long dimeric coiled-coil: the Acanthamoeba
myosin rod. Biochemistry 36:7876-7883.
[PubMed]
Carragher, B.O., Cheng, N., Wang, Z.-H., Korn, E.D.,
Reilein, A., Belnap, D., Hammer, III, J.A. and Steven,
A.C. (1998) Structural invariance of constitutively active
and inactive mutants of Acanthamoeba myosin IC
bound to F-actin in the rigor and ADP-bound states. Proc.
Natl. Acad. Sci. U.S.A. 95:15206-15211[PubMed]
Brzeska, H., Young, R., Knaus, U., and Korn, E.D.(1999)
Myosin I heavy chain kinase: cloning of the full-length
kinase and acidic lipid-dependent activation by Rac and
Cdc42. Proc. Natl. Acad. Sci. U.S.A.
96:394-399 [PubMed]
Redowicz, M.J., Hammer, III, J.A., Bowers, B., Zolkiewski,
M., Ginsburg, A., Korn, E.D., and Rau, D.C. (1999). Flexibility
of Acanthamoeba myosin rod minifilaments. Biochemistry
38:7243-7252 [PubMed]
Address
Edward D. Korn, Ph.D.
NIH, NHLBI, LCB
50 South Drive MSC 8017
Building 50, Room 2517
Bethesda, MD 20892-8017
Phone: (301) 496-1616
Fax: (301) 402-1519
E-mail: edk@nih.gov
Julie G. Donaldson: The ADP-ribosylation
factors (ARFs) are a family of GTP binding proteins that
regulate membrane traffic and organelle structure in the
cell . We are studying the function of ARF6, which affects
plasma membrane (PM) traffic and the actin cytoskeleton.
We have demonstrated that ARF6 regulates the movement of
PM into and out of a novel, endosomal recycling pathway
that influences cell surface morphology. The return of membrane
and ARF6 to the PM occurs at discrete sites along the peripheral
edges of cells and is associated with cortical actin polymerization
and the formation of protrusions. We have demonstrated that
this ARF6-regulated, membrane recycling pathway is required
during cell spreading and for the actin rearrangements and
membrane ruffling observed with activated forms of Rac1
GTPase. By contrast, ARF6-induced actin rearrangements occur
independently of any Rho proteins. The requirement for ARF6
activity for cell spreading, and Rac-induced ruffling implicates
the ARF6-regulated membrane recycling pathway as being a
critical component for cell shape alterations that may occur
during the cell cycle, cell migration, differentiation,
and metastasis. We are currently investigating potential
regulators of ARF6 activation and downstream targets which
mediate ARF6 function.
Selected recent publications:
Radhakrishna, H., Klausner, R.D., and Donaldson, J.G.
(1996) Aluminum fluoride stimulates cellular protrusions
in cells overexpressing the ARF6 GTPase., J. Cell Biol.,
134:935-947. [PubMed]
Radhakrishna, H., and Donaldson, J.G. (1997) ARF6 regulates
a novel plasma membrane recycling pathway. J. Cell Biol.,
139:49-61. [PubMed]
Song, J., Khachikian, Z., Radhakrishna, H., and Donaldson,
J.G. (1998) Localization of endogenous ARF6 to sites of
cortical actin rearrangement and involvement of ARF6 in
cell spreading. J. Cell Science, 111:2257-2267. [PubMed]
Radhakrishna, H., Al-Awar, O., Khachikian, Z., and Donaldson,
J. G. (1999) ARF6 requirement for Rac ruffling suggests
a role for membrane trafficking in cortical actin rearrangements.
J. Cell Science 112:855-866. [PubMed]
Address:
Julie G. Donaldson, Ph.D.
NIH, NHLBI, LCB
50 South Drive MSC 8017
Building 50, Room 2517
Bethesda, MD 20892-8017
Phone: (301) 402-2907
Fax: (301) 402-1519
E-mail: jdonalds@helix.nih.gov
Cellular Physiology Section:
Evan Eisenberg, M.D., Ph.D. and Lois Greene, Ph.D.
The 70 kDa class of heat shock proteins (Hsp70s) have been
termed molecular chaperones because they are required for
many crucial protein-protein interactions that occur in
the cell including protein folding, the formation and dissociation
of protein complexes, and the translocation of proteins
across intracellular membranes. To carry out these functions,
the Hsp70s not only hydrolyze ATP but also interact with
another class of molecular chaperones, the J-domain family,
which are required for recruitment of Hsp70s to their targets.
Such diverse proteins as DNA tumor-virus T antigen, interferon-induced
protein kinase inhibitor, and cysteine string proteins have
active J domains. Among the many functions of Hsp70 in the
cell, it plays a major role in endocytosis, removing clathrin
from clathrin-coated vesicles. We have discovered two unique
members of the J-domain family, one brain specific and one
occurring in all tissues, that are required for clathrin-uncoating
to occur. We are currently using site-directed mutagenesis,
knock-outs in both C. elegans and mice, and studies on GFP-clathrin
in cells to investigate the activity of these proteins in
endocytosis as well as NMR to study their structure. We
are also studying the role of Hsp70 and J-domain proteins
in the formation of steroid receptor complexes, in protein
folding, and in the protection of cells against stress e.g.
attack by the immune system.
Selected recent publications:
Greene, L.E., Zinner, R., Naficy, S., Eisenberg, E. (1995)
Effect of nucleotide on the binding of peptides to 70-kDa
heat shock protein. J. Biol. Chem. 270:2967-2973.
[PubMed]
King, C., Eisenberg, E., Greene, L. (1995) Polymerization
of 70-kDa heat shock protein by yeast DnaJ in ATP. J.
Biol. Chem. 270:22535-22540. [PubMed]
Ungewickell, E., Ungewickell, H., Holstein, S., Lindner,
R., Prasad, K., Barouch, W., Martin, B., Greene, L., and
Eisenberg, E. (1995) Role of auxilin in uncoating clathrin-coated
vesicles. Nature 378:632-635. [PubMed]
Jiang, R-F., Greener, T., Barouch, W., Greene, L. and
Eisenberg, E. (1997) Interaction of auxilin with the molecular
chaperone, Hsc70. J. Biol. Chem. 272:6141-6145.
[PubMed]
Rajapandi, T., Wu, Chengbiao, Eisenberg, E., and Greene,
L. (1998) Characterization of D10S and K71E mutants of
human cytosolic Hsp70. Biochemistry 37:7244-7250.
[PubMed]
Address:
Evan Eisenberg, M.D., Ph.D.
NIH, NHLBI, LCB
50 South Drive MSC 8017
Building 50, Room 2517
Bethesda, MD 20892-8017
Phone: (301) 496-2846
Fax: (301) 402-1519
E-mail: eisenbee@nih.gov
Membrane Enzymology Section:
Richard W. Hendler, Ph.D.
The interests of this section are focused on membrane-associated
enzymes and the role of the membrane in the activity of
the enzyme. Primary attention has been on integral membrane,
energy-transducing, proton pumps, namely cytochrome oxidase
and bacteriorhodopsin (BR). The experimental approach relies
heavily on computer-controlled procedures developed in our
laboratory to perform rapid kinetic optical measurements
which define steps in the enzyme-turnover and electrical
measurements to quantify energy-transducing events. With
cytochrome oxidase, we find that the electrons follow a
branched rather than a linear path from its redox centers
to O2. With BR, we have demonstrated a crucial
role for specific lipids of the membrane in the proton-pumping
photocycle and its regulation by actinic light. Work with
site-directed mutants is in progress to define the locus
of the lipid-protein interaction. Our studies with BR cover
purified BR preparations, BR-proteoliposomes, isolated cell
membrane fragments containing BR, and the intact cell. In
collaborations with other laboratories, we are trying to
correlate structural conformational states of the protein
with proton-pumping events using both FTIR and NMR.
Selected recent publications:
Mukhopadhyay, A. K., Dracheva, S., Bose,
S., and Hendler, R. W. (1996) Control of the integral
membrane proton pump, bacteriorhodopsin, by purple membrane
lipids of Halobacterium halobium. Biochemistry, 35:9245-9252.
[PubMed]
Barnett, S., Dracheva, S., Hendler, R.W.,
and Levin, I. (1996) Lipid-induced conformational changes
of an integral membrane protein: an infrared spectroscopic
study of the effects of Triton-X 100 treatment on the
purple membrane of Halobacterium halobium ET1001. Biochemistry,
35:4558-4567. [PubMed]
Bose, S., Hendler, R. W., Shrager, R. I.,
Chan, S. I., and Smith, P. D. (1997) Multichannel analysis
of single-turnover kinetics of cytochrome aa3 reduction
of O2. Biochemistry, 36:2439-2449. [PubMed]
Joshi, M. K., Dracheva, S., Mukhopadhyay,
A. K., Bose., and Hendler. R. W. (1998) Importance of
specific native lipids in controlling the photocycle of
bacteriorhodopsin. Biochemistry, 37:14463-14470. [PubMed]
Joshi, M. K., Bose, S., and Hendler, R.
W. (1999) Regulation of the Bacteriorhodopsin Photocycle
and Proton-Pumping in Whole Cells of Halobacterium salinarium.
Biochemistry, 38:8786-8793. [PubMed]
Address:
Richard Hendler, Ph.D.
NIH, NHLBI, LCB
50 South Drive MSC 8017
Building 50, Room 2517
Bethesda, MD 20892-8017
Phone: (301) 496-2610
Fax: (301) 402-1519
Email: rwh@helix.nih.gov
Molecular Cell Biology Section:
John A. Hammer, III, Ph.D.
Research
Interests
The long range goals of the section are to identify
and characterize unconventional myosins, and to define
their roles in the motility of cells and organelles.
Efforts focus primarily on Dictyostelium and mouse
as model systems. We use a variety of approaches to
attain these goals, including (i) biochemical and
biophysical characterization of purified or bacculovirus-expressed
myosins, (ii) localization of myosins using light
immunofluorescence microscopy, immumoelectron microscopy,
the expression of myosin/GFP chimeras, and subcellular
fractionation, (iii) identification of proteins that
interact with myosins using biochemical and molecular
genetic approaches, (iv) creation of cell lines/animials
that lack particular unconventional myosins using
homologous recombination/ antisense RNA expression,
and (v) characterization of these mutants using a
variety of cell biological assays, quantitative video
microscopy of cellular and intracellular motility,
and transmission electron microscopy. |
Mel-c melanocyte treated with cytochalasin and
stained for F-actin (blue), microtubules (red), and
the melanosome marker TRP-1 (green). |
| 
Aggregated Dictyostelium cells
stained for F-actin (red) and myosin IC (green). Yellow
indicates overlapping localization. |
Selected Recent Publications
Hammer, J.A. III, and Jung, G. (1996) The sequence of
the Dictyostelium myoJ heavy chain gene predicts a novel,
dimeric, unconventional myosin with a heavy chain molecular
mass of 258 kDa. J. Biol. Chem. 271:7120-7127. [PubMed]
Jung, G., Wu, X., and Hammer, J.A. III (1996) Dictyostelium
mutants lacking multiple classic myosin I isoforms reveal
combinations of shared and distinct functions. J. Cell
Biol. 122:305-323. [PubMed]
Wu, X., Wei, Q., Bowers, B., Kocher, R., and Hammer,
J.A. III (1997) Myosin V associates with melanosomes in
mouse melanocytes: Evidence that myosin V is an organelle
motor. J. Cell Sci. 110:847-859. [PubMed]
Wei, Q., Wu, X., and Hammer, J.A. III (1997) The predominant
defect in dilute melanocytes is in melanosome distribution
and not cell shape, supporting a role for myosin V in
melanosome transport. J. Muscle Res. Cell Motil. 18:517-527.
[PubMed]
Wu, X., Kocher, B., Wei, Q., and Hammer, J.A. III (1998)
Myosin Va associates with microtubule-rich domains in
both interphase and dividing cells. Cell Motil. Cytoskel.
40:286-303. [PubMed]
Wu, X., Bowers, B., Rao, K., Wei, Q., and Hammer, J.A.
III (1998) Visualization of melanosome dynamics within
wild type and dilute melanocytes suggests a paradigm for
myosin V function in vivo. J. Cell Biol. 143: 1899-1918.
[PubMed]
Wang, Z., Wang, F., Sellers, J., Korn, E.D., and Hammer,
J.A.III (1998) Analysis of the regulatory phosphorylation
site in Acanthamoeba myosin IC by using site-directed
mutagenesis. Proc. Natl. Acad. Sci. USA. 15200-15205.
[PubMed]
Submitted Paper
Jung, G., Remmert, K., Wu, X., Volosky, J.M., and Hammer,
J.A. III (2000) The Dictyostelium CARML Protein
Links Capping Protein and the Arp2/3 Complex to Type I
Myosins Through Their SH3 Domains. J. Cell Biology (Manuscript
#00-10-067)
Figure 9. The Appearance of Crowns in Wild Type
and p116 Cells. Vegetative Ax3 and p116-
cells were stained for actin, sectioned in 0.2 um intervals,
and the sections rendered in 3D. Shown are images at
30 and 90 degrees of title for Ax3 (Panels A and B)
and p116- (Panels C and D) cells. Arrowheads
point to the dorsal crowns. The mag bar is 3.4 um.
(View Movie in avi
format; requires the QuickTime
Viewer.)
Movies
Video Sequences of Melanosome Dynamics
Wu, X., Bowers, B., Rao, K., Wei, Q., and Hammer, J.A.
III (1998) Visualization of melanosome dynamics within
wild type and dilute melanocytes suggests a paradigm for
myosin V function in vivo. J. Cell Biol. 143 (7)
(Note: To view these movies you will need to download
the QuickTime
Viewer)
Sequence
Number |
Comments |
.mov file size |
| seq.#1 |
Melanosome movements in a heavily melanized
dilute melanocyte. |
574 KB |
| seq.#2 |
Melanosome movements in a lightly melanized
dilute melanocyte. |
470 KB |
| seq.#3 |
Melanosome movements within the thin
dendrite of a dilute melanocyte. |
235 KB |
| seq.#4 |
Melanosome movements within a large
dendritic extension of a dilute melanocyte. |
544 KB |
| seq.#5a |
A dilute melanocyte before the addition
of nocodazole (notice cell extension). |
882 KB |
| seq.#5b |
After the addition of nocodazole (the
same cell shown in seq#5a). |
194 KB |
| seq.#6a |
A dilute melanocyte before the addition
of nocodazole. (Notice cell center.) |
306 KB |
| seq.#6b |
After the addition of nocodazole (the
same cell shown in seq#6a). |
264 KB |
| seq.#7 |
30 minutes after nocodazole washout
(the same cell shown in seq#6b) |
779 KB |
| seq.#8 |
Melanosome dynamics within a well-spread
wild type melanocyte exhibiting an even distribution
of melanosomes. |
507 KB |
| seq.#9a |
Low mag image of melanosome dynamics
within a wild type melanocyte exhibiting a prominent
accumulation of melanosomes at the dendritic tip. |
909 KB |
| seq.#9b |
High mag image of melanosome dynamics
within the dendrite of the cell shown in seq.#9a. |
509 KB |
| seq.#9c1 |
Movements of individual melanosomes
within the dendrite shown in seq.#9b (centripetal). |
46 KB |
| seq.#9c2 |
Movements of individual melanosomes
within the dendrite shown in seq.#9b (centripetal). |
61 KB |
| seq.#9c3 |
Movements of individual melanosomes
within the dendrite shown in seq.#9b (centrifugal). |
61 KB |
| seq.#10a |
Low mag image of melanosome dynamics
within a wild type melanocyte exhibiting a prominent
accumulation of melanosomes along the edges of its
dendritic extension. |
762 KB |
| seq.#10b |
High mag image of melanosome dynamics
within a wild type melanocyte exhibiting a prominent
accumulation of melanosomes along the edges of its
dendritic extension. |
1009 KB |
| seq.#10c1 |
Movements of individual melanosomes
within the dendrite shown in seq.#10b (centripetal) |
157 KB |
| seq.#10c2 |
Movements of individual melanosomes
within the dendrite shown in seq.#10b (centripetal) |
101 KB |
| seq.#10c3 |
Movements of individual melanosomes
within the dendrite shown in seq.#10b (centripetal) |
66 KB |
| seq.#10c4 |
Movements of individual melanosomes
within the dendrite shown in seq.#10b (centrifugal). |
51 KB |
| seq.#10c5 |
Movements of individual melanosomes
within the dendrite shown in seq.#10b (centrifugal). |
36 KB |
| seq.#11 |
Generation of a dilute-like phenotype
by expression of the myosin Va tail domain. |
1930 KB |
| seq.#12a |
Melanosome dynamics in microtubule-depleted
wild type melanocytes. |
307 KB |
| seq.#12b |
Melanosome dynamics in mirotubule-depleted
dilute melanocytes. |
302 KB |
Links
Address
John A. Hammer, III
NIH, NHLBI, LCB
50 South Drive MSC 8017
Building 50, Room 2517
Bethesda, MD 20892-8017
Phone: (301) 496-8960
Fax: (301) 402-1519
E-mail: hammerj@fido.nhlbi.nih.gov
Last updated: April 16
, 2001
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