Myosin-X and myosin-XIX in intracellular transport

Myosin motor proteins travel along actin filaments and power the movements responsible for force generation, vesicle and organelle trafficking, and formation of protrusive cellular structures. This dissertation discusses myosin‐X and the novel myosin‐XIX. Myosin‐X is a MyTH4‐FERM myosin that localizes to the tips of the slender, actin‐based cellular protrusions known as filopodia. Moreover, it undergoes intrafilopodial motility and is a master regulator of filopodia formation. Previously, we demonstrated that through its FERM domain, myosin‐X binds to β‐integrins. Here we describe experiments designed to elucidate the functional consequence of the myosin‐ X‐β‐integrin interaction. We report that: 1) knock‐down of myosin‐X inhibits cell spreading and adhesion, 2) the FERM domain of myosin‐X, unlike that of talin, does not activate integrins, and 3) myosin‐X co‐transports with integrins in filopodia. These results support a model in which myosin‐X is a component of the filopodia tip complex and participates in the intrafilopodial transport of other tip proteins. Myosin‐XIX is the founding member of a novel class of myosins and is the last uncharacterized human myosin. Here we report that myosin‐XIX is expressed in many cell types and shows striking localization to mitochondria. Additionally, over‐expression of a GFP‐tagged myosin‐XIX results in a dramatic gain of function phenotype in which mitochondria become hyper‐motile. To reveal the endogenous function of myosin‐XIX, we designed and tested a series of siRNAs against myosin‐XIX. With this tool, we can further characterize the function of myosin‐XIX, the first vertebrate myosin shown to localize to the mitochondria. These studies advance our understanding of the role of myosin‐X in filopodia and provide the first report of the mitochondria‐associated myosin‐XIX

Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and Caveolin-1

Abstract Background In the current study we investigated the role of caveolin-1 (cav-1) in pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-1 is the major structural protein in caveolae; small Ω-shaped invaginations within the plasma membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding protein to organize multiple molecular complexes regulating a variety of cellular events. Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and metastasis; however, the molecular mechanisms have not been described. The small monomeric GTPases are among several molecules which associate with cav-1. Classically, the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion. RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif. Results Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines derived from primary tumors and low expression in those derived from metastases. Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation. Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression. Conclusion Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.http://deepblue.lib.umich.edu/bitstream/2027.42/112733/1/12943_2005_Article_110.pd

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals

Human Myo19 Is a Novel Myosin that Associates with Mitochondria

Mitochondria are pleomorphic organelles [1, 2] that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease [3-5]. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport [6]. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues and antibodies to human Myo19 detect a ∼109kD band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain-of-function where the majority of the mitochondria move continuously. Moving mitochondria travel for many microns with an obvious leading end and distorted shape. The motility and shape-change are sensitive to latrunculin B, indicating that both are actin-dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells

Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and caveolin-1

© 2005 Lin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

The dendritic and T cell responses to herpes simplex virus-1 are modulated by dietary vitamin E

Previous studies from our laboratory have shown that dietary α-tocopherol (vitamin EVE) is essential for regulating the cytokine and chemokine response in the brain to herpes simplex virus-1 (HSV-1) infection. The timing of T cell infiltration is critical to the resolution of central nervous system HSV-1 infections. Specifically, the appearance of “neuroprotective” CD8+IFN-γ+ T cells is crucial. During CNS infection, CD8+ T cell priming and expansion in the draining lymph node, followed by recruitment and expansion occurs in the spleen with subsequent accumulation in the brain. Weanling male BALB/cByJ mice were placed on VE deficient (Def) or adequate (Adq) diets for 4 weeks followed by intranasal infection with HSV-1. VE Def mice had fewer CD8+IFN-γ+ T cells trafficking to the brain despite increased CD8+IFN-γ+ T cells and activated dendritic cells in the periphery. VE Def mice had increased T regulatory cells in the periphery and brain and the increase in Tregs decreases CD8+ T cell numbers in the brain. Our results demonstrate that adequate levels of VE are important for trafficking antigen-specific T cells to the brain and dietary VE levels modulate T regulatory and dendritic cells in the periphery