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 major research goal is to elucidate the mechanisms that underlie directed cell migration from its initiation to its termination. This interest stems from the pivotal role of migration in a variety of normal and pathological processes extending from the development to the adult. 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. In this context, it is no surprise 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 contributes pivotally to the formation of tumors, which requires the invasion of vasculature 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.
Current research projects include: a) the assembly and disassembly of adhesions, b) the trafficking of adhesion components, c) characterizing the migration proteome d) developing in vivo systems for studying migration e) developing new imaging technologies for migration studies, and f) studying mechanisms of synapse formation and spine dynamics. While most of our studies utilize migrating fibroblasts and neuronal cells, we are interested in any aspect of migration - from embryonic development to cancer and regeneration.
I. The assembly of adhesions
Our goal is to determine the sequence of events that lead to the formation of the adhesions and identify the molecules and mechanisms that regulate them. We are approaching this using ratio analysis of cells expressing two different fusion proteins, e.g., CFP/YFP or other fluorescent pairs, to determine their relative kinetics of incorporation into adhesions. In parallel, we are identifying the molecules that regulate assembly using cell lines with putative regulatory genes knocked out or knocked down (RNAi)/, expressing dominant negative constructs, or treated with specific inhibitors. We have found that the kinetics by which some components enter adhesions can be clearly dissected and placed into a temporal (and perhaps causal) sequence. Other components enter with indistinguishable kinetics. We have no evidence suggesting that smaller adhesion assemblies coalesce to form larger adhesions. From our data thus far, we have concluded that assembly follows, at least in part, a nucleated, sequential model with some components entering in preformed complexes.
II. Adhesion disassembly and turnover
Using an approach similar to that described above for adhesive assembly, we are studying the disassembly of adhesions. Adhesion disassembly occurs at two locations in the cell - behind the leading edge and at the cell rear. The former is a relatively new observation that we have made and is coupled to the formation of protrusions. We have developed sensitive, quantitative assays of disassembly. Our goal is to use ratio imaging to determine the sequence of steps that lead to the breakdown of adhesions and identify the molecules and mechanisms that regulate it. Using inhibitors and cell lines that do not express putative regulatory molecules, 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. Our observations suggest that adhesion disassembly is not the simple reversal of assembly, but instead, appears more concerted. We are also interested in the mechanism that leads to the disassembly of adhesions. For example, are the affinities of the associations among adhesion molecules reduced or is their organization altered due to altered contractility? We are addressing these questions using patterned photobleaching and image correlation techniques (see below).
III. Trafficking of adhesive components
Adhesions is to serve as traction points over which cells move during migration. This results in the relocation of adhesion components in the front to the rear. This results in the removal of adhesion components from the cell front and their accumulation at the rear. We are interested in whether trafficking mechanisms serve to move material from the rear and replenish them at the cell front. We find that vesicular integrins traffic from the cell front and rear toward the perinuclear region. We also see some integrins moving in vesicles from the perinuclear region to the base of the protrusion. More interestingly, we have evidence that some signaling molecules, like GIT1/PAK, traffic to and from adhesions in large macromolecular complexes rather than in vesicles. These observations raise several important questions. 1)What is the nature of the perinuclear compartment; is it a recycling compartment with its components trafficked to the cell front or is it a lysosomal compartment? 2) What are the relative contributions of biosynthesis and recycling to delivering material toward the leading edge? 3) Do vesicular integrins traffic alone or in complexes with other molecules? 4) What molecules traffic in non-vesicular complexes? What determines the polarity and pathway of these trafficking phenomena? We are approaching these questions in several ways, including photoactivation, pattern photobleaching, correlation microscopy (see below), and selective inhibitions of vesicle trafficking of GFP tagged integrins and other adhesion molecules.
IV. The migration proteome
We are engaged in two complementary activities devoted to identifying novel migration related molecules and characterizing the migration proteome. One is based on expression cloning, using cDNA libraries fused to GFP. The approach is to identify novel proteins that localize in adhesions or migration related structures, perturb adhesion or cytoskeletal organization, or promote migration. We have already identified several novel genes. One is a microtubule-associated protein, GLFND, that is involved in microtubule organization. Another is GIT1, a protein that was originally identified by its binding to a G-protein coupled receptor kinase. This protein contains an ARF-GAP domain that regulates vesicle trafficking and perhaps Rac activity and has binding sites for paxillin and Pix (an exchange factor for Rac and PAK binding protein). It localizes in adhesions, the leading edges, and in cytoplasmic signaling complexes and functions by locally activating the Rac signaling complex leading to the formation of protrusions. Recently, we have shown that GIT1 is expressed prominently in neurons, where it resides in synapses and regulates the formation of spines and synapses via its Pix/Rac/PAK binding properties. We are continuing to screen for and characterize novel molecules.
The second proteomic project uses tandem mass spectrometry. The goal is to identify novel proteins, create an interaction map using co-immunopurification strategies, and characterize all the phosphorylation sites. This collaborative project has already identified a number of interesting new proteins and identified several novel phosphorylation sites.
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 very 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. One goal is to study the nature of the migration pathway and the factors that regulate it. Another goal is to characterize the molecular events, i.e., localization, dynamics, and function of migration related molecules that regulate migration in vivo.
We have developed several systems for these kinds of studies. They include: migrations of a) muscle precursors from the somites to the limb, b) neuronal precursors along the rostral migratory stream from the subventricar zone to the olfactory bulb, c) migration of interneurons in the developing spinal cord, and d) tangential migrations of neuronal precursors in the cerebellum. Unlike cells migrating in vitro, those in vivo reveal a prominent long protrusion and highly directed migration pathways. The protrusion appears to result, in part, from the local activation of Rac, which also determines the direction of migration. DCC and ADAMS family members also appear to contribute to directionality.
Our immediate objectives are to elucidate the mechanism by which ADAMS family members contribute to directional migration and to elucidate the mechanisms that lead to highly polarized protrusions. Our approaches include using activation specific antibodies to determine the polarity of molecules in cells migrating in situ. We also plan to develop transgenic mice with endogenous genes replaced by GFP fusions to study molecular dynamics during migration along with mice in which key genes are either knocked down or knocked out.
VI. Image Correlation Microscopy
Fluctuation methodologies, e.g., image correlation and correlation microscopy, are technologies that are only beginning to be applied to cell biological questions. In brief, these technologies allow us to measure the absolute concentrations, degrees of aggregation, and rates of flow and diffusion of cellular components at high spatial resolution. When two differently labeled species are present, it allows measurement of their association, including concentrations of interacting species and dynamics. Our present focus is on image correlation microscopy, since it can be used retrospectively to determine changes in aggregation, dynamics, concentration, etc. across the cell during transient processes like signaling, adhesion formation or disassembly, and the extension or retraction of a protrusion. In addition to estimating flow rates, image correlation techniques also measure the direction of movement. Thus image correlation can provide spatial maps of dynamics and concentration across the cell and allow us to determine how they change over time. Using labeled integrin, a-actinin, and paxillin, we have recently found spatial differences in dynamics, interactions between a-actinin and integrin (in both static and dynamic complexes), and observed changes in aggregation state as adhesions disassemble. The quantitative measurements of concentrations, binding constants, and kinetic parameters will also be used to develop models of adhesion assembly. We also are using this technology to monitor the activation of signaling molecules by measuring interactions with their target molecules.
VII.Synapse formation
While not generally studied in this context, synapse formation can be considered as a migration endpoint. Growth cone movement terminates as synapses form. In addition, the dendritic spines present at synapses are actin rich sites that recapitulate many migratory phenomena including movement in response to stimulation. 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 locally regulate Rac activity. Our observations appear to provide an explanation for some human mutations that produce nonsyndromic mental retardation. We plan to develop these studies further by identifying the signaling pathways that regulate synapse formation and the role of adhesive and Rho family signaling in this process.
Selected References:
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)
