Search PubMed⌕ Search

Biomedical subjects

Alan F Horwitz

Publications and source records attributed to Alan F Horwitz.

At least 19 recordsLinked to original sources
PubMed2007

alpha5 integrin signaling regulates the formation of spines and synapses in hippocampal neurons.

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.

Animals↗
PubMed2006

Mechanism of polarized protrusion formation on neuronal precursors migrating in the developing chicken cerebellum.

Directed cell migration results from the polarization of the cellular motile apparatus by integration of extracellular signals, which are presented in a three-dimensional, spatiotemporal manner in living organisms. To investigate the mechanism underlying the highly polarized and directional nature of migration in vivo, we have developed an imaging system for observing rhombic lip cell migration in the developing chicken cerebellum. First, we show that Cdc42 is the central regulator of the overall polarity, morphology and protrusion formation in these cells. However, perturbation of canonical polarity effectors of Cdc42, e.g. the Par6-Par3-aPKC complex, does not disrupt the cell asymmetry, whereas it affects orientation of the tip of the leading process. In contrast to Cdc42, Rac is required for the generation of protrusions but not the overall polarity. Function interference of class IA phosphoinositide 3-kinase abrogates both directional extension and maintenance of the long leading process, whereas PTEN modulates the size of the protrusion. Actomyosin contractility is important for coordinated spreading of the tip of the leading process in situ. Finally, ErbB4 functions in the generation of protrusions on the rhombic lip cells. These results suggest that polarized protrusion formation on neuronal precursors may occur by a more divergent and complex mechanism than that seen in studies of other cell types growing on planar substrates.

Actomyosin↗
PubMed2006

From imaging to understanding: Frontiers in Live Cell Imaging, Bethesda, MD, April 19-21, 2006.

A recent meeting entitled Frontiers in Live Cell Imaging was attended by more than 400 cell biologists, physicists, chemists, mathematicians, and engineers. Unlike typical special topics meetings, which bring together investigators in a defined field primarily to review recent progress, the purpose of this meeting was to promote cross-disciplinary interactions by introducing emerging methods on the one hand and important biological applications on the other. The goal was to turn live cell imaging from a "technique" used in cell biology into a new exploratory science that combines a number of research fields.

Animals↗
PubMed2006

Phosphorylation of serine 709 in GIT1 regulates protrusive activity in cells.

G protein-coupled receptor kinase-interacting protein (GIT)1 is a multidomain, adaptor protein that regulates cellular processes, such as migration and protrusive activity, by bringing together various signaling molecules, including PIX, PAK, and paxillin. Mutants of GIT1, which lack the C-terminal paxillin binding domain, fail to mediate its effects on migration and protrusions, suggesting that sites within this domain are critical to GIT1 function. In this study, we show that serine 709, which is located within the paxillin binding domain, regulates GIT1 function. Phosphorylation of serine 709 is necessary for GIT1-induced effects on protrusions. Phosphorylation of this site also regulates GIT1 interaction with paxillin, which could serve to target GIT1 to the leading edge of cells. As shown by an in vitro kinase assay, PAK phosphorylates GIT1 on serine 709. Taken together, our results indicate that GIT1 phosphorylation on serine 709 increases its binding to paxillin and regulates protrusive activity in cells.

Adaptor Proteins, Signal Transducing↗
PubMed2006

In vivo dynamics of Rac-membrane interactions.

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.

3T3 Cells↗
PubMed2006

Identification of protein networks associated with the PAK1-betaPIX-GIT1-paxillin signaling complex by mass spectrometry.

The process of cell motility involves coordinate signaling events among proteins associated in interactive integrin-linked networks. Mass spectrometric analysis of immunoprecipitation-derived protein mixtures have provided efficient means of identifying proteomes. In this study, we investigate strategies to enhance the detection of interactome proteins for the known signaling module: PAK1, betaPIX, GIT1, and paxillin. Our results indicate that near-endogenous expression levels of bait protein enhances the identification of associated proteins, and that phosphatase inhibition augments the detection of specific protein interactions. Following the analysis of a large pool of spectral data, we have identified and mapped clusters of proteins that either share common interactions among the four bait proteins of interest or are exclusive to single bait proteins. Taken together, these data indicate that biochemical manipulations can enhance the ability for LC-MS/MS to identify interactome proteins, and that qualitative screening of multiple samples leads to the compilation of proteins associated with a known plexus.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Migration of perilesional microglia after focal brain injury and modulation by CC chemokine receptor 5: an in situ time-lapse confocal imaging study.

Microglia rapidly become reactive in response to diverse stimuli and are thought to be prominent participants in the pathophysiology of both acute injury and chronic neurological diseases. However, mature microglial reactions to a focal lesion have not been characterized dynamically in adult vertebrate tissue. Here, we present a detailed analysis of long-distance perilesional microglial migration using time-lapse confocal microscopy in acutely isolated living slices from adult brain-injured mice. Extensive migration of perilesional microglia was apparent by 24 h after injury and peaked at 3 d. Average instantaneous migration speeds of approximately 5 microm/min and peak speeds >10 microm/min were observed. Collective, directed migration toward the lesion edge was not observed as might be expected in the presence of chemoattractive gradients. Rather, migration was autonomous and could be modeled as a random walk. Pharmacological blockade of the cysteine-cysteine chemokine receptor 5 reduced migration velocity and the number of perilesional migratory microglia without affecting directional persistence, suggesting a novel role for chemokines in modulation of discrete migratory parameters. Finally, activated microglia in the denervated hippocampal stratum oriens did not migrate extensively, whereas human immunodeficiency virus-1 tat-activated microglia migrated nearly twice as fast as those at the stab lesion, indicating a nonuniform microglial response to different stimuli. Understanding the characteristics and specific molecular mechanisms underlying microglial migration after neural injury could reveal novel targets for therapeutic strategies for modulating neuroinflammation in human diseases.

Amides↗
PubMed2005

A GIT1/PIX/Rac/PAK signaling module regulates spine morphogenesis and synapse formation through MLC.

Three of seven recently identified genes mutated in nonsyndromic mental retardation are involved in Rho family signaling. Two of the gene products, alpha-p-21-activated kinase (PAK) interacting exchange factor (alphaPIX) and PAK3, form a complex with the synaptic adaptor protein G-protein-coupled receptor kinase-interacting protein 1 (GIT1). Using an RNA interference approach, we show that GIT1 is critical for spine and synapse formation. We also show that Rac is locally activated in dendritic spines using fluorescence resonance energy transfer. This local activation of Rac is regulated by PIX, a Rac guanine nucleotide exchange factor. PAK1 and PAK3 serve as downstream effectors of Rac in regulating spine and synapse formation. Active PAK promotes the formation of spines and dendritic protrusions, which correlates with an increase in the number of excitatory synapses. These effects are dependent on the kinase activity of PAK, and PAK functions through phosphorylating myosin II regulatory light chain (MLC). Activated MLC causes an increase in dendritic spine and synapse formation, whereas inhibiting myosin ATPase activity results in decreased spine and synapse formation. Finally, both activated PAK and activated MLC can rescue the defects of GIT1 knockdown, suggesting that PAK and MLC are downstream of GIT1 in regulating spine and synapse formation. Our results point to a signaling complex, consisting of GIT1, PIX, Rac, and PAK, that plays an essential role in the regulation of dendritic spine and synapse formation and provides a potential mechanism by which alphaPIX and PAK3 mutations affect cognitive functions in mental retardation.

Animals↗
PubMed2005

Infiltrative microgliosis: activation and long-distance migration of subependymal microglia following periventricular insults.

BACKGROUND: Subventricular microglia (SVMs) are positioned at the interface of the cerebrospinal fluid and brain parenchyma and may play a role in periventricular inflammatory reactions. However, SVMs have not been previously investigated in detail due to the lack of a specific methodology for their study exclusive of deeper parenchymal microglia. METHODS: We have developed and characterized a novel model for the investigation of subventricular microglial reactions in mice using intracerebroventricular (ICV) injection of high-dose rhodamine dyes. Dynamic studies using timelapse confocal microscopy in situ complemented the histopathological analysis. RESULTS: We demonstrate that high-dose ICV rhodamine dye injection resulted in selective uptake by the ependyma and ependymal death within hours. Phagocytosis of ependymal debris by activated SVMs was evident by 1d as demonstrated by the appearance of rhodamine-positive SVMs. In the absence of further manipulation, labelled SVMs remained in the subventricular space. However, these cells exhibited the ability to migrate several hundred microns into the parenchyma towards a deafferentation injury of the hippocampus. This "infiltrative microgliosis" was verified in situ using timelapse confocal microscopy. Finally, supporting the disease relevance of this event, the triad of ependymal cell death, SVM activation, and infiltrative microgliosis was recapitulated by a single ICV injection of HIV-1 tat protein. CONCLUSIONS: Subependymal microglia exhibit robust activation and migration in periventricular inflammatory responses. Further study of this population of microglia may provide insight into neurological diseases with tendencies to involve the ventricular system and periventricular tissues.

Journal Article↗
PubMed2005

Cell migration: an overview.

Cell migration is an essential process for normal development and homeostasis that can also contribute to important pathologies. Not surprisingly, there is considerable interest in understanding migration on a molecular level, but this is a difficult task. However, technologies are rapidly emerging to address the major intellectual challenges associated with migration. In this chapter, we outline the basics of cell migration with an emphasis on the diverse systems, methodologies, and techniques described in this book. From the contributions presented, it is apparent that the next few years should produce major advances in our understanding of cell migration.

Animals↗
PubMed2004

FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.

Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Talin: an emerging focal point of adhesion dynamics.

The adhesion protein talin and the phosphoinositide PIP2 are emerging as key modulators of adhesion dynamics. Recent genetic studies on talin demonstrate its physiological role in organizing adhesions, stabilizing integrin-actin linkages and mediating integrin signaling in vivo. Biophysical force measurements provide further evidence that it is required for the reinforcement of the extracellular matrix-integrin-actin connection. Knockdown data along with structural analyses establish a major role for talin in 'inside-out' integrin activation through its direct interaction with integrin cytoplasmic domains. A recently uncovered role for talin is the recruitment of a PIPKI gamma isoform to adhesions. This introduces a novel connection between talin and PIP2 generation. Finally, PIP2 also stimulates the transient, direct binding interaction of the Arp2/3 complex with vinculin and thus may couple adhesion to actin assembly.

Animals↗
PubMed2003

Synapse formation is regulated by the signaling adaptor GIT1.

Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

Actins↗
PubMed2003

Illuminating adhesion complexes in migrating cells: moving toward a bright future.

Cell migration is a complex, tightly regulated process that involves the continuous formation and disassembly of adhesions. Despite the importance of these processes, very little is known about the factors that regulate adhesion dynamics during migration. Recent advances in imaging technologies are allowing monitoring of these processes during migration and are providing insight into the mechanisms that regulate them.

Animals↗
PubMed2002

Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream.

Precursors of the olfactory interneurons migrate from the subventricular zone via the rostral migratory stream (RMS). To investigate the molecular mechanisms by which RMS cells migrate, we used a slice preparation, which allows the migrating cells to be imaged at very high temporal and spatial resolution in the presence of added inhibitors. Using immunohistochemistry, we first determined that the alpha1-, beta8-, and beta1-integrin subunits and the alpha5- and gamma1-laminin subunits are expressed during embryonic day 16 to the early postnatal stage. During early postnatal days, alpha(v)- and beta6-integrins appeared, and their expression persisted throughout adulthood. The migrating cells also expressed the netrin receptors neogenin and Deleted in Colorectal Carcinoma (DCC). Netrin-1 is expressed in olfactory mitral cells. Anti-integrin antibodies inhibited the production of protrusions as well as cellular translocation. In contrast, anti-DCC antibodies primarily altered the direction of the protrusions; consequently, the migration was no longer unidirectional, and the speed was reduced. Thus, the interaction of DCC, possibly through an interaction with netrin-1, contributes to the direction of migration by regulating the formation of directed protrusions. In contrast, the integrins function in production of protrusions and cellular translocation, with different integrins participating at different developmental stages.

Aging↗