Wound healing with electric potential.
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Cell Movement↗
Biomedical subjects
Alan Rick Horwitz
Publications and source records attributed to Alan Rick Horwitz.
Probing the integrin-actin linkage using high-resolution protein velocity mapping.
Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins alpha-actinin, alpha5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at alpha-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.
3T3 Cells↗
Integrating adhesion, protrusion, and contraction during cell migration.
Cell migration is fastest when the strength of the adhesion between the cell and the substrate is neither too strong nor too weak. In this issue of Cell, reveal how adhesion and cytoskeletal dynamics are integrated to optimize migration speed.
Actins↗
Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics.
Continuous adhesion formation and disassembly (adhesion turnover) in the protrusions of migrating cells is regulated by unclear mechanisms. We show that p21-activated kinase (PAK)-induced phosphorylation of serine 273 in paxillin is a critical regulator of this turnover. Paxillin-S273 phosphorylation dramatically increases migration, protrusion, and adhesion turnover by increasing paxillin-GIT1 binding and promoting the localization of a GIT1-PIX-PAK signaling module near the leading edge. Mutants that interfere with the formation of this ternary module abrogate the effects of paxillin-S273 phosphorylation. PAK-dependent paxillin-S273 phosphorylation functions in a positive-feedback loop, as active PAK, active Rac, and myosin II activity are all downstream effectors of this turnover pathway. Finally, our studies led us to identify in highly motile cells a class of small adhesions that reside near the leading edge, turnover in 20-30 s, and resemble those seen with paxillin-S273 phosphorylation. These adhesions appear to be regulated by the GIT1-PIX-PAK module near the leading edge.
Amino Acid Sequence↗
Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions.
Migrating cells can sustain a relatively constant direction of lamellipodial protrusion and locomotion over timescales ranging from minutes to hours. However, individual waves of lamellipodial extension occur over much shorter characteristic times. Little understanding exists regarding how cells might integrate biophysical processes across these disparate timescales to control the directional persistence of locomotion. We address this issue by examining the effects of epidermal growth factor (EGF) stimulation on long-timescale directional persistence and short-timescale lamellipodial dynamics of EGF receptor-transfected Chinese hamster ovary cells migrating on fibronectin-coated substrata. Addition of EGF increased persistence, with the magnitude of increase correlating with fibronectin coating concentration. Kymographic analysis of EGF-stimulated lamellipodial dynamics revealed that the temporal stability of lamellipodial protrusions similarly increased with fibronectin concentration. A soluble RGD peptide competitor reduced both the persistence of long-timescale cell paths and the stability of short-timescale membrane protrusions, indicating that cell-substratum adhesion concomitantly influences lamellipodial dynamics and directional persistence. These results reveal the importance of adhesion strength in regulating the directional motility of cells and suggest that the short-timescale kinetics of adhesion complex formation may play a key role in modulating directional persistence over much longer timescales.
Animals↗
Cell migration: integrating signals from front to back.
Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis; contributes to tissue repair and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. The migrating cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes. This review describes the mechanisms underlying the major steps of migration and the signaling pathways that regulate them, and outlines recent advances investigating the nature of polarity in migrating cells and the pathways that establish it.
Actin Cytoskeleton↗
Building bridges through collaboration--a pathway for interdisciplinary research.
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Biology↗
Identification of a novel microtubule-associated protein that regulates microtubule organization and cytokinesis by using a GFP-screening strategy.
Microtubules play critical roles in a variety of cell processes, including mitosis, organelle transport, adhesion and migration, and the maintenance of cell polarity. Microtubule-associated proteins (MAPs) regulate the dynamic organization and stability of microtubules, often through either cell-specific or cell division stage-specific interactions. To identify novel cytoskeletal-associated proteins and peptides that regulate microtubules and other cytoskeletal and adhesive structures, we have developed a GFP cDNA screening strategy based on identifying gene products that localize to these structures. Using this approach, we have identified a novel MAP, GLFND, that shows homology to the Opitz syndrome gene product [6], localizes to a subpopulation of microtubules that are acetylated, and protects microtubules from depolymerization with nocodazole. Expression of an N-terminal deletion binds microtubules but alters their organization. During the cell cycle, GLFND dissociates from microtubules at the beginning of mitosis and then reassociates at cytokinesis. Furthermore, ectopic expression of GLFND inhibits cell division and cytokinesis in CHO cells. These observations make GLFND unique among MAPs characterized thus far.
Animals↗
Breaking barriers through collaboration: the example of the Cell Migration Consortium.
Understanding complex integrated biological processes, such as cell migration, requires interdisciplinary approaches. The Cell Migration Consortium, funded by a Large-Scale Collaborative Project Award from the National Institute of General Medical Science, develops and disseminates new technologies, data, reagents, and shared information to a wide audience. The development and operation of this Consortium may provide useful insights for those who plan similarly large-scale, interdisciplinary approaches.
Cell Movement↗
GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration.
GIT1 is a multidomain protein that is thought to function as an integrator of signaling pathways controlling vesicle trafficking, adhesion and cytoskeletal organization. It regulates ARF GTPases and has binding domains for paxillin and PIX, which is a PAK-binding protein and an exchange factor for Rac. We show that GIT1 cycles between at least three distinct subcellular compartments, including adhesion-like structures, the leading edge and cytoplasmic complexes. The cytoplasmic structures, which also contain paxillin, PAK and PIX, do not detectably co-localize with endosomal Golgi or membrane markers, suggesting that they represent a novel supramolecular complex. The GIT1 cytoplasmic complexes are motile and tended to move toward the cell periphery where they joined existing adhesions. In retracting regions of the cells, the GIT1 complexes moved away from the disassembling adhesions toward the cell body. Using deletion mutants, we have identified domains that target GIT1 to each of the compartments. Localization to adhesions and the leading edge requires the paxillin-binding domain, which comprises the C-terminal 140 residues (cGIT1), whereas targeting to the cytoplasmic complexes requires the central region that contains ankyrin repeats and the PIX-binding domain. Expression of GIT1 or cGIT, but not nGIT1 in which the paxillin-binding domain is deleted, increases the rate of migration and the size and number of protrusions. The latter are inhibited when GIT1 is co-expressed with a kinase-dead PAK, suggesting that the GIT1 interaction with PAK is required for enhanced migration and protrusive activity. Furthermore, GIT1 targets constitutively activated PAK to adhesions and the leading edge via its interaction with paxillin. Since expression of cGIT targets endogenous GIT1 to the leading edge, it appears that the leading edge is the location of GIT1 responsible for these activities. Thus, GIT1 is a component of a motile, multimolecular complex that traffics a set of signaling components to specific locations in the cell where they regulate localized activities.
Animals↗