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Research Interests

The Horwitz Lab resides in the Department of Cell Biology at the University of Virginia in Charlottesville, VA. The lab is also affiliated with the Department of Microbiology and the Neuroscience Program and is a member of the Cell Migration Consortium.

Our research goals are to elucidate the mechanisms that underlie directed cell migration and dendritic spine morphogenesis. The migration interest stems from its pivotal role in a such a wide variety of normal and pathological processes; they extend from embryonic development to tissue regeneration, immune surveillance, and cancer. During development, for example, cells migrate from their birthplaces to distant locations where they then differentiate. While this process is repeated throughout the embryo, it plays out spectacularly in the nervous system. Neuronal precursors migrate from their birthplaces to their final residences and then proceed to extend neuronal growth cones to their targets, where they form synaptic connections with appropriate target cells. It is no surprise, therefore, that a large fraction of the congenital brain (and heart defects) arise from perturbed cell migration. Migration contributes to numerous pathological phenomena as well. It plays a pivotal role in the formation of tumors, which requires the invasion of blood vessels, as well as in metastasis, the spread of tumors from the primary tumor mass to distant sites where secondary tumors form. Migration also contributes to other disease processes including chronic inflammatory diseases, via leukocyte invasion, and vascular disease via smooth muscle migration. Finally, migration participates centrally in normal tissue regeneration and wound repair and is an issue in successful stem cell transplantation.

Another research goal is the mechanism and regulation of synapse formation and dendritic spine morphogenesis in hippocampal neurons, which is thought to mediate synaptic plasticity in learning and memory. We view the post synaptic structure as a specialized adhesion and spine morphogenesis as related to the actin dynamics and organization that drives cell migration. With this analogy, we translate our understanding of migration to derive insights into synapse formation and dendritic spine dynamics.

Adhesion, migration, signaling, imaging, and the cytoskeleton are the common themes in all of our research. Current research projects include: a) the mechanisms of adhesion assembly, disassembly and signaling, b) mechanisms by which cells polarize, c) developing new imaging technologies for migration studies, d) migration in vivo, and f) mechanisms of synapse formation and dendritic spine dynamics. Our studies employ a variety of cell types and models, and we are interested in any aspect of migration - from embryonic development to cancer and regeneration. We take an eclectic approach to our research and use a number of methodologies including: state of the art live cell imaging, total internal reflection (TIRF), confocal, correlation, and single molecule methods, mass spectrometry based proteomics, transgenic animals, and more standard biochemical, cell biologic and molecular genetic methods.

I. The assembly and disassembly of adhesions

Adhesions at the cell front stabilize the protruding regions, serve as traction points for migration, and produce signals that regulate migration. Adhesions near the front edge of protruding regions form and disassemble (turnover) continuously - a process that is linked to protrusion itself. The signaling through these adhesions is modulated by tension, which inhibits adhesion turnover, signaling, and protrusion (a type of mechanotransduction). Our goal is to determine the sequence of events by which these adhesions assemble and identify the molecules and mechanisms that regulate it. We are approaching this using phospho-proteomics (using mass spectrometry in collaboration with the Hunt lab), ratio analysis of cells expressing two different fusion proteins, e.g., GFP/mCherry or other fluorescent pairs, and correlation microscopy. In parallel, we are identifying the molecules that regulate assembly.

Using single molecule sensitivity and high speed total internal reflection microscopy (TIRF) microscopy, we have shown that nascent adhesions form in the lamellipodium, near the leading edge of migrating cells. These adhesions contain many components common to other adhesions, and they all appear to enter the adhesion simultaneously suggesting they reside in preformed complexes or enter by diffusion in response to a common signal. These “nascent” adhesions are fixed in space and stable until they reach the lamellipodium/lamellum interface, where they either elongate and mature or disassemble as the actin reorganizes. The formation of nascent adhesions requires actin polymerization, which we hypothesize initiates and nucleates the process, and their turnover is driven by uncoupling from actin. The inter-relation between the organization and polymerization of actin on adhesion assembly and disassembly is a major interest.

Nascent adhesions mature by growth and elongation along a-actinin/actin templates. Knockdown of a-actinin reveals its critical role in organizing the lamellipodium, actin filaments and adhesions. Adhesion components assemble along the template sequentially suggesting a stepwise mechanism for adhesion growth and elongation. The critical role and regulation of a-actinin in adhesion maturation and lamellipodium formation is a subject of continued interest. Myosin II is an end point of signaling pathways that regulate adhesion formation. We have found, however, that nascent adhesions form and turnover in the absence of myosin II. Instead, it is their elongation and growth (maturation) that requires myosin II. Surprisingly, the crosslinking properties of myosin II appear to drive the early stages, rather than contraction, per se. Along with adhesion maturation, myosin II also determines migration rates. We are testing the hypothesis that differences in myosin activity determine differences in the migration rates and adhesion properties of different cell types including cancer cells. Leukocytes, for example, are highly motile and use a specific integrin for their migration. We are using different cell types and integrins to reveal the role of myosin in these differences.

We are also probing the mechanism of adhesion formation and turnover using correlation microscopy, which measures clustering, binding affinities, and interactions of adhesion molecules (see below). Our initial results suggest that adhesions form from addition of monomers, or very small complexes, rather than incorporation of large, pre-formed multi-molecular complexes. The mechanism by which initial adhesions nucleate, i.e., integrin clustering and activation or binding to dendritic actin, and the specific associations that drive adhesion formation are among our major interests. Our observations using correlation microscopy also suggest that adhesion disassembly is not a simple reversal of assembly, but instead, appears to involve the release of large multimolecular complexes. We are also interested in the mechanisms that underlie adhesion disassembly.

II. Adhesive signaling

We have identified several molecules that regulate adhesion disassembly, including FAK, Src, and paxillin. We have also identified important targets of these molecules and defined the outlines of a regulatory pathway that controls adhesion turnover. We have identified phosphorylation sites on paxillin, for example, serve as a switch that regulates adhesion turnover and maturation. One of these sites recruits a GIT1/Pix/Pak/Rac signaling module to adhesions near the leading edge where it localizes Rac activation, which drives actin polymerization and adhesion formation. We are pursuing the synergy among the phosphorylation sites on paxillin as well as the mechanisms by which they regulate Rac activity.

Our observation that myosin II clustering drives adhesion maturation has potential implications for the mechanisms of mechanotransduction. One of our goals is to examine the signaling properties of different classes of adhesions and the role of tension and clustering in these signals. In addition, the profound role of myosin activity and key adhesion-related signaling switches suggests interesting approaches to defining invasive signatures in tumors.

III. Correlation and fluctuation microscopy

Fluorescence correlation and other fluctuation-based microscopies use spatial and temporal fluctuations in the fluorescence of tagged molecules to determine local concentration, aggregation, diffusion, binding and unbinding, and transport. When used with two different molecules, each with a different color, the same parameters can be obtained for interacting complexes, when they exist. Originally, these measurements were made in a single locus on the cell. In collaboration with the Enrico Gratton and Paul Wiseman, we have greatly expanded this methodology and can now develop cellular maps of the above parameters, at high spatial and temporal resolution.

While this technology is still new, it has already produced important, new data and ideas. They include: 1) the existence of a molecular “clutch” that links integrins to actin retrograde flow. The clutch is regulated and only partially efficient, with points of “slippage” in the linkage. 2) Adhesions appear to form largely from the rapid incorporation of monomers or small complexes. They disassemble by the release of large multi-molecular complexes. 3) Adhesions slide by a treadmilling mechanism. Our goal is to use fluctuation methods to develop a quantitative molecular model for adhesion assembly and disassembly. We also are using this technology to monitor the activation of signaling molecules by measuring interactions with their target molecules. We plan to extend correlation methods to cells migrating in three dimensions and in vivo.

IV. Mechanisms by which migrating cells polarize

The formation of a define cellular front and rear, front-rear polarization, is critical for directed cell migration. While microtubules, Cdc42, and PI3 kinase are all implicated in establishing this polarity, the underlying mechanism remains unclear. We have found that knocking down myosin IIB eliminates front-rear polarity, as well as the positioning of the Gogi and organization of microtubules. Furthermore, activated myosin IIB creates an extended tail and increases directed migration. Finally, varying expression of myosin isoforms, seen in different cell types and cancer cells, creates differences in polarity and migration. The dynamics of myosin isoforms in migrating cells and organization of actin in cells with differing levels of polarization all point to the organization of acto-myosin filaments and adhesions in establishing polarity. Our working hypothesis is that myosin IIB establishes the rear by creating large actin bundles and stable adhesions rather than the dynamic adhesions that support protrusion at the front. We are studying the kinases that activate MLC at the rear, and determining how myosin IIB organizes the microtubules and the Golgi apparatus. Finally, we are interested in the role of different isoforms in cancer invasion.

V. In vivo migration

Most studies of cell migration utilize cells growing on tissue culture dishes. While there are many advantages to studying migration in such a defined environment, it differs significantly from the environment in which cells migrate in living organisms. In vivo, for example, the substrate and growth factors are three dimensional and complex. We have developed slice technology which allows us to study migration of cells in situ. This is an optically accessible system that retains essentially all of the properties of the in vivo environment including directional migration. We have developed several in vitro systems including migration of neuronal precursors, immune cells in the brain in response to injury, tumor cells in the brain, and muscle precursors in the trunk. Our goal is to characterize the molecular events, i.e., localization, dynamics, and function of migration related molecules that regulate and drive migration in vivo. The migration of many cells is not integrin based, particularly in the brain. Another goal is to study the mechanisms of non integrin based migration.

VII. Dendritic spines and synapse formation

Synaptic plasticity and learning are commonly linked to alterations in dendritic spine morphology and dynamics. Dendritic spines and the formation of synapses have much in common with migrating cells. We are pursuing this parallel by translating our findings on migration to spine morphology and synapse formation . Using hippocampal neurons in culture, we found that GIT1 localizes in synapses where it functions to organize a Rac/PAK/Pix signaling module that serves to localize Rac activity in spines and thereby mediate morphogenesis. These observations provide an explanation for human mutations that produce nonsyndromic mental retardation. We are now studying the signaling pathway that connects NMDA receptor activity to GIT1 and myosin activation and determining how actomyosin filaments dictate spine morphology and synaptic plasticity.

Selected References:

Vicente-Manzanares M, Koach MA, Whitmore L, Lamers ML, Horwitz AF (2008) Segregation and activation of myosin IIB creates a rear in migrating cells. Journal of Cell Biology. 183(3): 543-54. Abstract (Pubmed)

Choi CK, Vicente-Manzanares M, Zareno J, Whitmore L, Mogilner A, Horwitz AF (2008) Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor independent manner. Nature Cell Bio. 10: 1039-1050. Abstract (Pubmed)

Digman MA, Brown CM, Horwitz AF, Mantulin WW, Gratton E (2007) Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy. Biophys J. 94(7):2819-31. Abstract (Pubmed)

Mayhew MW, Jeffery ED, Sherman NE, Nelson K, Polefrone JM, Pratt SJ, Shabanowitz J, Parsons JT, Fox JW, Hunt DF, Horwitz AF (2007) Identification of phosphorylation sites in BetaPIX and PAK1. J. Cell Science. 120: 3911-3918. Abstract (Pubmed)

Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz AF (2007) Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells.176(5): 573-80. Abstract (Pubmed)

Mayhew MW, Webb DJ, Kovalenko M, Whitmore L, Fox JW, Horwitz AF (2006) Identification of Protein Networks Associated with the PAK1-BetaPIX-GIT1-Paxillin Signaling Complex by Mass Spectrometry. J of Proteome Research.5(9): 2417-2423. Abstract (Pubmed)

Sakakibara A and Horwitz AF (2006) Mechanism of polarized protrusion formation on neuronal precursors migrating in the developing chicken cerebellum. J. Cell Science. 119(Pt 17): 3583-92. Abstract (Pubmed)

Nayal A, Webb DJ, Brown CM, Schaefer EM, Vicente-Manzanares M, Horwitz AR (2006) Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics. J Cell Biol. 173(4): 587-9. Abstract (Pubmed)

Wiseman PW, Brown CM, Webb DJ, Herbert B, Johnson NL, Squier JA, Ellisman MH, Horwitz AF (2004) Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microcopy. J Cell Science. 117(Pt 23): 5521-5534. Abstract (PubMed)

Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion diassembly. Nat Cell Biol. 6(2): 154-161. Abstract (PubMed)

Murase S and Horwitz AF (2004) Directions in cell migration along the rostral migratory stream: the pathway for migration in the brain. Curr Top Dev Biol. 61: 135-152. No Abstract Available.

Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science. 1704-1709. Abstract (PubMed)

Zhang H, Webb DJ, Asmussen H, Horwitz AF (2003) Synapse formation is regulated by the signaling adaptor GIT1. J Cell Biol. 161: 131-42. Abstract (PubMed)

Manabe R, Whitmore L, Weiss JM, Horwitz AR (2002) Identification of a Novel Microtubule-Associated Protein that Regulates Microtubule Organization and Cytokinesis by Using a GFP-Screening Strategy. Curr Biol. 12: 1946-51. Abstract (PubMed)

Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells -- over and over and over again. Nat Cell Biol. 4(4): E97-100. Abstract (PubMed)

Murase S and Horwitz AF (2002) Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream. J Neurosci. 22:3568-79. Abstract (PubMed)

Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells -- over and over and over again. Nat Cell Biol. 4(4): E97-100. Abstract (PubMed)

Webb DJ, Asmussen H, Murase S, Horwitz AF (2002) Cell migration in slice cultures. Methods Cell Biol. 69: 341-58. No Abstract Available.

Manabe, et al (2002) GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration. J Cell Sci.115:1497-510. No Abstract Available.

Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells -- over and over and over again. Nat Cell Biol. 4(4): E97-100. Abstract (PubMed)

Laukaitis CM, Webb DJ, Donais K, Horwitz AF (2001) Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells. J Cell Biol. 153(7): 1427-40. Abstract (PubMed)

West KA, Zhang H, Brown MC, Nikolopoulos SN, Riedy MC, Horwitz AF, Turner CE (2001) The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL). J Cell Biol. 154(1): 161-76. Abstract (PubMed)

Knight B, Laukaitis C, Akhtar N, Hotchin NA, Edlund M, Horwitz AR (2000) Visualizing muscle cell migration in situ. Curr Biol. 10(10): 576-85. Abstract (Pubmed)

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