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Cell Motility

How do cells assemble, organize their shape, move, and target molecules to specific intracellular locations? Motility is essential for nearly all cell processes.

Research on cellular motility is centered at The Pennsylvania Muscle Institute (PMI) , an interdisciplinary group of research investigators focused on understanding the mechanisms and regulation of motile biological systems. Research is conducted using biophysics, biochemistry, genetics, physiology and ultrastructure to understand cell migration, intracellular transport, smooth, cardiac and skeletal muscle contraction, the structure/function relationship of molecular motors, chromosome segregation, cell division, muscle cell development, myofibril assembly and gene therapy targeted to muscle. The PMI is prominent in technological and methodological development for these investigations especially in advanced light microscopy, structural spectroscopy, photochemical reagents, image processing, and molecular biology. Motility research and graduate and post-doctoral research opportunities span the following areas:

The Institute also enhances the research and intellectual environment at the University of Pennsylvania through its Seminar Program, Research Retreats, Journal Clubs and Core Facilities. The PMI was founded in 1973 is supported by the University of Pennsylvania School of Medicine, the National Institutes of Health, and Private Foundations.

Cell Migration and Intracellular Transport

Cell migration and intracellular transport are complex cellular processes that require a dynamic cytoskeleton (actin and microtubules) and molecular motors (myosin, kinesin, and dynein).

Cell migration includes whole-cell locomotion and the regulation of the cell shape and extracellular attachment. Cell migration is crucial for several normal and pathological processes, including: cell and tissue development, wound healing, immune response, and metastases of tumors.

Intracellular transport is the movement and targeting of vesicles and proteins to specific cellular regions. Sometimes this transport occurs over long distances, like down the nerve axon, and sometimes this transport is simply the movement of a vesicle through the cell cortex. Transport also includes the proper delivery and localization of organelles (e.g., mitochondria).

Members of the Pennsylvania Muscle Institute are using and developing state-of-the-art techniques to understand the molecular events required for the regulation of cell migration and intracellular transport.

Mechanism and Regulation of Muscle Contraction

Three types of muscles, skeletal, cardiac and smooth, convert metabolic energy to mechanical force and work for myriad functions in the organism. The wide impact of understanding the molecular mechanism and control of muscle contraction in cell biology should be emphasized. The molecular mechanism of muscle contraction is similar to that of cellular and organelle motility which are prerequisites for normal cell development and function. Contractility is also a model for biological energy transduction and enzymology in general. The relationship between the amino-acid sequence, structural biology and functional output of motor proteins and contractile proteins are being studied intensively in many PMI labs.

As with non-muscle motility, the general paradigm cells use for generating motion is a motor protein sliding along a linear filamentous track. This architecture was first discovered in muscle cells because these elegant machines express and assemble concentrated and highly periodic interdigitating arrays of myosin and actin filaments that are particularly amenable to structural, mechanical and biochemical studies. The filaments do not change length when the muscle contracts, but they slide relative to each other using energy liberated by hydrolysis of ATP to ADP and phosphate. The relationships between the biochemical steps of ATP hydrolysis, the mechanical development of force and filament sliding and the molecular structural changes are the major goals of this research.

State-of-the-art technologies, such as laser photolysis of 'caged' substrates and signaling molecules, time-resolved electron microscopy, optical traps (laser tweezers), fluorescence polarization spectroscopy and molecular genetics are applied to discover the mechanism of chemical-to-mechanical energy conversion.

Control of muscle contraction takes many forms, including calcium signaling, phosphorylation by specific kinases and receptor-effector signal cascades. As with contraction, signaling and control of muscle cells act as prardigms for corresponding processes throughout cell biology. Modern biophysical and molecular biological techniques are being applied to detail the molecular mechanisms of these pathways. PMI laboratories are prominent in developing new techniques with broad impact.

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Structure/Function Studies of Molecular Motors

Molecular motors (dynein, kinesin, and myosin) are Nature's engineering masterpieces. These remarkable proteins use chemical energy (ATP) to generate mechanical force and motion, and in doing so, play key roles in nearly every physiological process. Muscle contraction is one of the most dramatic examples of motor function.

PMI members are determining - at the atomic level - how molecular motors use ATP to generate motility. Members use a combination of techniques to determine the ATP-induced structural changes in the motor that lead to force generation. These techniques include single-molecule spectroscopy, single-molecule force measurements, heterologous expression of mutant molecular motors, and transient biochemical kinetic measurements.

Chromosome Segregation and Cell Division

Major events in the cell cycle must be coordinated with each other to ensure the equal partitioning of genetic information from the parental cell to the two progeny cells. Among these events are chromosome segregation and cytokinesis, two fundamental processes that must be regulated both temporally and spatially. Microtubules and their associated motor proteins function together to exert the force that moves the chromosomes but how they do this is not well understood. In addition, the molecular mechanisms by which the spindle integrity is checked before cell division are far from clear. Cytokinesis is also carried out by a dynamic structure, the actomyosin contraction system. How the type II myosins and F-actin interact with each other to generate the contractile force is not known. Furthermore, the signal specifying the cell division site as well as the cell cycle signal triggering actin ring formation and contraction still remain to be identified and characterized. All these questions are being addressed in a variety of systems by several members of the PMI.

Gene Therapy and Neuromuscular Disease

PMI members investigate several muscle-based pathologies, including muscular dystrophy, inherited cardiomyopathies, and other neuromuscular diseases. The goal is to understand the molecular basis of these diseases, allowing for the development of effective therapies. A recent area of research concentration of PMI laboratories is the use of viral-mediated gene transfer to study many aspects of muscle function. The primary application of this tool has been gene therapy for diseases in muscle and other tissues. In this context, missing or mutant genes can be replaced by the introduction and expression of a correct gene. These efforts have focused on the treatment of muscular dystrophy. However, because gene transfer is very efficient in muscle, other studies have utilized skeletal muscle as a "factory" for generating proteins needed elsewhere in the body.

In addition to the therapeutic applications of gene transfer, viral-mediated gene transfer is being used to understand properties of muscle contraction, adaptation, and energetics. By overexpressing both normal and mutant genes in adult muscle, one can perturb the conditions of muscle after development has taken place. These studies include the expression of 1) contractile proteins such as Troponin C, 2) growth factors such as IGF-I and myostatin, and 3) enzymes which can attenuate ischemic-reperfusion injury, such as catalase and arginine kinase.

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Muscle Cell Development and Myofibril Assembly

During vertebrate embryogenesis, skeletal muscle cell differentiation is coordinated, spatially and temporally, with the development of the embryonic body plan by cell-cell signaling between the axially-located neural tube and newly forming somites. These interactions result in an ordered series of changes in gene expression from the synthesis of myogenic regulatory factors, synthesis and assembly of muscle proteins into myofibrils and isoform changes of the major contractile proteins. Much less is known about the developmental steps leading to the differentiation of cardiac muscle cells.

Myofibrillogenesis, one of the major steps in the early development of skeletal and cardiac muscles, occurs in an orchestrated series of events involving the formation of actin and myosin filaments, their assembly into an interdigitating array and their interaction with cross-linking proteins to form the sarcomere. We still know very little about the roles played by individual sarcomeric proteins in the assembly of myofibrils. New techniques of molecular biology and genetics combined with advances in biophysical and imaging techniques provide tools for examining this problem that were not possible in the past.

This program is to use such a multidisciplinary approach combining the techniques of cell biology, biophysics, genetics and molecular biology to discover important steps in the differentiation of precursor cells into skeletal and cardiac muscle cells and the parameters governing the assembly of myofibrils.

Advanced Technological Development

The development of new technologies, motivated by significant biological problems is a major emphasis of the Institute. Imaging technology coupled to advanced video-enhanced light microscopy, confocal microscopy, infrared optical traps (laser tweezers) quantitative and spatially-resolved fluorescence detection for protein orientation and mobility and for ion-sensitive probes, localized laser photolysis of ‘caged’ molecules and digital image processing are emerging technologies with wide potential application. The Institute’s efforts have resulted in the design and fabrication of novel instrumentation for biological electron and X-ray spectroscopy, in the synthesis and utilization of ‘caged’ compounds for laser photolysis studies of transient events associated with triggering of muscle contraction, time resolved polarization spectrophotometry and novel uses of fluorescently labeled proteins to study the assembly of filamentous systems in living cells. The PMI operates a Light Microscopy Core Facility for development of advanced techniques and an Optical Trapping and Protein Manipulation core facility under NIH Program Project Grant support. A new, highly capable multi-wavelength confocal microscope is supported by an NSF Shared Instrumentation Grant.

A very recent area of research concentration is nanotechnology. The Institute’s involvement has been in development of techniques to manipulate and study single functioning protein molecules. In many enzyme and polynucleotide systems, important elementary events at the molecular level are obscured within the average behavior of a molecular population. Studying enzymes one at a time has only recently become technically feasible but new information, not otherwise available, can be gained from such an approach. Work in this area will allow, for the first time, measurements of time-resolved structural information, forces, elasticity and molecular displacements on single biomolecules.

The immediate interest in developing these new techniques is for investigating the molecular mechanisms underlying motor protein-based cell motility and muscle contraction. However these methods are applicable to all enzymes that undergo structural changes between compact domains during their functional activity. The PMI will serve as a resource center for single molecule nanotechnology studies of enzyme systems outside cell motility, welcoming other investigators to study their proteins of interest by these powerful methods.

For more information on the Pennsylvania Muscle Institute, and the investigators involved in the above programs, please visit http://www.med.upenn.edu/~pmi/

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Biological Engineering Network at the University of Pennsylvania
1010 Vagelos Research Labs / 3340 Smith Walk / Philadelphia PA 19104-6383
tel. 215-573-6813 ~ fax. 215-573-6815 ~ e-mail: ben-penn@pobox.upenn.edu