Synapse formation; Regulation of gene expression in nerves and muscle; Membrane protein
biogenesis and localization; Gene expression and regulation
..
An important problem in Cellular and Molecular Neurobiology concerns the mechanisms
whereby
electrically-excitable cells regulate the expression and localization of proteins involved
in signal
transduction during development and differentiation. Research in my laboratory focuses on
the
regulation of membrane protein biogenesis in skeletal muscle fibers with emphasis on
molecules such as acetylcholinesterase (AChE) involved in synaptic transmission. Synaptic
components such as AChE are highly concentrated at the vertebrate neuromuscular junction
and other cholinergic synapses in the central and peripheral nervous systems.
Understanding how these molecules are regulated will reveal many of the general mechanisms
that regulate molecules at synapses. Our overall objectives are to determine the relative
contributions of electrical activity and neural influences on the transcriptional,
translational, and post translational control of AChE. In addition, these molecules also
serve as markers for the development of the synaptic basal lamina, and for the mechanisms
involved in targeting specific molecules to synapses.
Compartmentalization in Skeletal Muscle Fibers:
We have previously shown that transcription, translation, and assembly of AChE is
compartmentalized in multinucleated skeletal muscle fibers. Our experiments provided
evidence that
muscle cells are functionally divided into compartments, encompassing the regions
surrounding
individual nuclei, in contrast to earlier notions that muscle mRNAs were free to diffuse
and randomly translated throughout the cell. These studies provided the physical basis for
postulating that locally transcribed and translated membrane proteins would be selectively
transported and localized to specialized regions of the cell surface overlying the nucleus
of origin, such as occurs at the neuromuscular synapse. Subsequent experiments in my
laboratory indicated that specialized regions of the plasma membrane form directly over
myotube nuclei and in fact over 90% of synaptic protein clusters form within one nuclear
diameter (15-20 µm) of the underlying nucleus. In vivo, innervated regions of muscle
fibers contain more AChE mRNA than the neighboring noninnervated regions, approximately
500-1000 copies of the AChE mRNA per nucleus. Therefore a very selective and specific
mechanism exists for localizing synaptic components to the cell surface overlying the
nucleus of origin. This mechanism begins with localized transcription, translation, and
assembly of the synaptic components inside the cell and ends with the selective targeting
and retention of these molecules on the overlying membrane.
..
Assembly and Localization of Acetylcholinesterase:
A related area of research focuses on the assembly of the multiple oligomeric forms of
AChE and
their targeting to different kinds of synapses in the central and peripheral nervous
systems. Although
a single gene encodes all AChE forms, different classes of post-translational
modifications are
involved in sorting and attaching the AChE to specific extracellular sites. In one set of
published
experiments, we transplanted avian AChE molecules on the frog neuromuscular synapse,
thereby
demonstrating the existence of specific receptors at sites of nerve-muscle contact. We are
now in the
process of identifying the "receptors" involved. One of these receptors has now
been identified as
perlecan, a heparan sulfate proteoglycan which is concentrated at the neuromuscular
junction.
Our current research emphasizes the signal transduction systems linking changes in
membrane
depolarization to changes in gene expression in muscle, the mechanisms responsible for
localizing
AChE in electrically excitable cells, and the molecular events leading to the assembly of
functional
AChE molecules in the rough endoplasmic reticulum. These studies employ combinations of
many
techniques including direct and indirect immunofluorescence, in situ hybridization,
quantitative
PCR, construction and expression of chimeric proteins in heterologous cell systems, and
many
other approaches in Cell and Molecular Biology.
Paul Cohen Postdoctoral Fellowship, Muscular Dystrophy Association, 1977-1978
Fellow of the Muscular Dystrophy Association, 1978-1980
Alfred P. Sloan Research Fellow in Neuroscience, 1982-1985
NIH Research Career Development Award, 1988-1993
J. Cell Biology Editorial Board, Assistant Editor, 1989-1995
NIH Neurological Sciences I Study Section, regular member, 7/90-6/94
Hebrew University Visiting Professor of Medicine and Science, Jerusalem, Israel, Spring
1995
École Normale Supérieur, Paris, France, Spring 1998
Rossi SG and Rotundo RL. (1993) Localization of "Non Extractable"
Acetylcholinesterase to the Vertebrate Neuromuscular Junction. J Biol. Chem. 268, 19152-19159
Jasmin BJ, Lee RK and Rotundo RL. (1993) Compartmentalization of Acetylcholinesterase mRNA
and Enzyme at the Vertebrate Neuromuscular Junction. Neuron 11, 467-477.
Rotundo, R.L., Rossi, S.G., Godinho, R.O., Vazquez, A.E., and Trivedi, B. (1995)
Restricted Regulation of Acetylcholinesterase Transcription, Translation, and
Localization. In: Enzymes of the Cholinesterase Family, Quinn D.M., Balasubramanian, A.S.,
Doctor, B.P., and Taylor, P. (eds). Plenum Press, New York.
Rossi, S.R. and Rotundo, R.L. (1996) Transient Interactions Between Collagen-Tailed
Acetylcholinesterase and Sulfated Proteoglycans Prior to Immobilization on the
Extracellular Matrix. J. Biol. Chem. 271:
1979-1987.
Rotundo, R.L., Rossi, S.G., and Anglister, L. (1997) Transplantation of Quail
Collagen-tailed Acetylcholinesterase Molecules on to the Frog Neuromuscular Synapse J. Cell Biol. 136: 367-374.
Lee, R.K., Jasmin, B.J., and Rotundo, R.L. (1998) Development of the Neuromuscular
Junction. In: Principles of Medical Biology. Edward E. Bittar (Ed.). JAI Press, Greenwich,
CT.
Rotundo, R.L., Rossi, S.G., and Peng, H.B., Targeting Acetylcholinesterase Molecules to
the Neuromuscular Synapse. J. Physiol. (Paris) 92:
195-198 (1998).
Peng, H.B., Xie, H., Rossi, S.G., and Rotundo, R.L., Acetylcholinesterase Clustering at
the Neuromuscular Junction Involves Perlecan and Dystroglycan. J. Cell Biol. 145: 911-921 (1999).
Rossi, S.G., Vazquez, A.E., and Rotundo, R.L., Local Control of Acetylcholinesterase Gene
Expression in Multinucleated Skeletal Muscle Fibers. (J. Neurosci., 2000, in press).
Rossi, S.R. and Rotundo, R.L., Transcriptional Control of Acetylcholinesterase Expression
During Muscle Differentiation (in preparation).
Rossi, S.G., Vazquez, A.E., and Rotundo, R.L., Local Control of Acetylcholinesterase Gene
Expression in Multinucleated Skeletal Muscle Fibers. J. Neurosci. 20: 919-928 (2000).
Adams, M.E., Kramarcy, N.,Krall, S.P., Rossi, S.G., Rotundo, R.L., Sealock, R., and
Froehner, S.C., Absence of "-Syntrophin Leads to Structurally Aberrant Neuromuscular
Synapses Deficient in Utrophin. J. Cell Biol. 150: 1385-1397, (2000).
Jacobson, C., Côté, P., Rossi, S.G., Rotundo, R.L., and Carbonetto, S.. The Dystroglycan
Complex is Necessary for Stabilization of Acetylcholine Receptor Clusters at Neuromuscular
Junctions and Formation of the Synaptic Basal Lamina. J. Cell Biol. (Submitted).
Drapeau, P., Buss, R.R., Ali, D.W., Legendre, P., and Rotundo, R.L.. Synaptic Organization
Limits the Development of Fast Neuromuscular Transmission. (submitted to J. Neuroscience)
N.I.H. (R01-AG05917)(7/1994-6/1999), R. Rotundo, P.I.
"Regulation of Acetylcholinesterase Synthesis and Assembly"
N.I.H. SBIR (R43 HL59944) (9/97-8/98), A. Haces, P.I., R. Rotundo, Co-P.I.
"Polycationic Lipids for Transfection of Biomolecules"
N.I.H. (R01-AG05917)(7/1999-6/2004), R. Rotundo, P.I.
"Regulation of Acetylcholinesterase Synthesis and Assembly"
Muscular Dystrophy Association (7/1/99-6/30/02), R. Rotundo, P.I.
"Assembly of Acetylcholinesterase and the Synaptic Basal Lamina"
Richard L. Rotundo, Ph.D.
Department of Cell Biology and Anatomy (R-124)
University of Miami School of Medicine
1600 N.W. 10th Avenue
Miami, Florida 33136
Tel: (305) 243-6940
Fax: (305) 545-7166
E-mail: rrotundo@molbio.med.miami.edu
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