Leanna Whitmore

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.

Actin and |[alpha]|-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner

Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an α-actinin–actin template that elongates centripetally from nascent adhesions. α-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.

Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells

We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front–back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front–back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.

Segregation and activation of myosin IIB creates a rear in migrating cells

We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB–enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.

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 α-actinin, α5-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 α-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.

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front–back polarity in migrating cells

Myosin IIA and IIB synergistically generate front–back polarity through their effects on actomyosin bundling formation and stability, and adhesion maturation, which are mediated by localized Rac GEF depletion.

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity.

ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity, while ROCK2 regulates contractile force, Rac1 activity, and cofilin-mediated actin remodeling at the leading edge of migratory cells and the spine head of neurons.

Identification of phosphorylation sites in GIT1.

G protein-coupled receptor kinaseinteracting protein 1 (GIT1) was originally identified as an ADP ribosylation factor GTPase-activating protein (ARF-GAP) that binds Gprotein-coupled receptor kinases (GRKs) and regulates membrane trafficking (Premont et al., 1998). Subsequent studies have shown a much broader function for GIT1 and GIT2/PKL as regulators of migrationrelated processes, including adhesion and cytoskeletal organization (Manabe et al., 2002; Mazaki et al., 2001; West et al., 2001; Zhao et al., 2000). GIT function and localization are most likely mediated through its interaction with various signaling molecules, including paxillin, p21-activated kinase interacting exchange factor (PIX), focal adhesion kinase (FAK), phospholipase C (PLC ) and mitogen-activated protein kinase kinase 1 (MEK1) (Bagrodia et al., 1999; Haendeler et al., 2003; Manabe et al., 2002; West et al., 2001; Yin et al., 2004; Zhao et al., 2000). In fibroblasts and epithelial cells, GIT1 regulates migration and protrusive activity by assembling and targeting multi-protein signaling complexes that contain actin regulators, such as PIX and the Rac/Cdc42 effector p21-activated kinase (PAK), to adhesions and the leading edge of a protrusion (Di Cesare et al., 2000; Manabe et al., 2002). Another GIT family member, PKL, which is the chicken homolog of GIT2, also recruits PIX and PAK to adhesions through its interaction with paxillin (Brown et al., 2002). Once in adhesions, GIT1 promotes their disassembly through a PIX-dependent mechanism and stimulates motility (Zhao et al., 2000).

A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front–back polarity

A group of phosphorylatable serine residues within the nonhelical domain of NMII-B controls the ability of NMII-B to generate stable migratory front–rear polarity.

Gestational Changes in the Germinal Matrix of the Normal Rhesus Monkey Fetus

ABSTRACT: To explain the reported predisposition to germinal matrix hemorrhage in premature infants, pathogenetically important morphological features of the germinal matrix should be present in the 3rd trimester and rapidly change near term. Such features were sought in this study of the germinal matrix and its vasculature in normal rhesus monkey fetuses. The matrix cells, glia, ependyma, and capillaries showed no important structural changes during the 3rd trimester. The terminal vein tributaries were greatly enlarged by 148 days, but cellular and collagen support in their walls was minimal at this time. The latter features developed by the final days of gestation. These findings do not support a structural immaturity or specialization of the germinal matrix predisposing to germinal matrix hemorrhage. Our results, therefore, support the recent emphasis on physiological parameters in the pathogenesis and prevention of germinal matrix hemorrhage.