Myosin-X: a MyTH-FERM myosin at the tips of filopodia

Myosin-X (Myo10) is an unconventional myosin with MyTH4-FERM domains that is best known for its striking localization to the tips of filopodia and its ability to induce filopodia. Although the head domain of Myo10 enables it to function as an actin-based motor, its tail contains binding sites for several molecules with central roles in cell biology, including phosphatidylinositol (3,4,5)-trisphosphate, microtubules and integrins. Myo10 also undergoes fascinating long-range movements within filopodia, which appear to represent a newly recognized system of transport. Myo10 is also unusual in that it is a myosin with important roles in the spindle, a microtubule-based structure. Exciting new studies have begun to reveal the structure and single-molecule properties of this intriguing myosin, as well as its mechanisms of regulation and induction of filopodia. At the cellular and organismal level, growing evidence demonstrates that Myo10 has crucial functions in numerous processes ranging from invadopodia formation to cell migration

Myosin-X and disease

Myosin-X (Myo10) is a motor protein well known for its role in filopodia formation. New research implicates Myo10 in a number of disease states including cancer metastasis and pathogen infection. This review focuses on these developments with emphasis on the emerging roles of Myo10 in formation of cancer cell protrusions and metastasis. A number of aggressive cancers show high levels of Myo10 expression and knockdown of Myo10 has been shown to dramatically limit cancer cell motility in 2D and 3D systems. Myo10 knockdown also limits spread of intracellular pathogens marburgvirus and Shigella flexneri. Consideration is given to how these properties might arise and potential paths of future research

Compact Nuclei in Galaxies at Moderate Redshift:II. Their Nature and Implications for the AGN Luminosity Function

This study explores the space density and properties of active galaxies to z=0.8. We have investigated the frequency and nature of unresolved nuclei in galaxies at moderate redshift as indicators of nuclear activity such as Active Galactic Nuclei (AGN) or starbursts. Candidates are selected by fitting imaged galaxies with multi-component models using maximum likelihood estimate techniques to determine the best model fit. We select those galaxies requiring an unresolved point-source component in the galaxy nucleus, in addition to a disk and/or bulge component, to adequately model the galaxy light. We have searched 70 WFPC2 images primarily from the Medium Deep Survey for galaxies containing compact nuclei. In our survey of 1033 galaxies, the fraction containing an unresolved nuclear component greater than 5% of the total galaxy light is 9+/-1% corrected for incompleteness. In this second of two papers in this series, we discuss the nature of the compact nuclei and their hosts. We present the upper limit luminosity function (LF) for low-luminosity AGN (LLAGN) in two redshift bins to z=0.8. Mild number density evolution is detected for nuclei at -18 -16 and this flatness, combined with the increase in number density, is inconsistent with pure luminosity evolution. Based on the amount of density evolution observed for these objects, we find that almost all present-day spiral galaxies could have hosted a LLAGN at some point in their lives. We also comment on the likely contribution of these compact nuclei to the soft X-ray background.Comment: 50 pages, 14 figures, to appear in ApJ, April 199

Visualization of individual carbon nanotubes with fluorescence microscopy using conventional fluorophores

We demonstrate that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast in fluorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolar solvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction. The reverse-contrast images also provide clear indication of nanotube location. © 2003 American Institute of Physics

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile , c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability

Headless Myo10 Is a Negative Regulator of Full-length Myo10 and Inhibits Axon Outgrowth in Cortical Neurons

Myo10 is an unconventional myosin that localizes to and induces filopodia, structures that are critical for growing axons. In addition to the ∼240-kDa full-length Myo10, brain expresses a ∼165 kDa isoform that lacks a functional motor domain and is known as headless Myo10. We and others have hypothesized that headless Myo10 acts as an endogenous dominant negative of full-length Myo10, but this hypothesis has not been tested, and the function of headless Myo10 remains unknown. We find that cortical neurons express both headless and full-length Myo10 and report the first isoform-specific localization of Myo10 in brain, which shows enrichment of headless Myo10 in regions of proliferating and migrating cells, including the embryonic ventricular zone and the postnatal rostral migratory stream. We also find that headless and full-length Myo10 are expressed in embryonic and neuronal stem cells. To directly test the function of headless and full-length Myo10, we used RNAi specific to each isoform in mouse cortical neuron cultures. Knockdown of full-length Myo10 reduces axon outgrowth, whereas knockdown of headless Myo10 increases axon outgrowth. To test whether headless Myo10 antagonizes full-length Myo10, we coexpressed both isoforms in COS-7 cells, which revealed that headless Myo10 suppresses the filopodia-inducing activity of full-length Myo10. Together, these results demonstrate that headless Myo10 can function as a negative regulator of full-length Myo10 and that the two isoforms of Myo10 have opposing roles in axon outgrowth

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production During Stationary Phase

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability

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

Cdc42 and ARP2/3-independent regulation of filopodia by an integral membrane lipid-phosphatase-related protein

Filopodia are dynamic cell surface protrusions that are required for proper cellular development and function. We report that the integral membrane protein lipid-phosphatase-related protein 1 (LPR1) localizes to and promotes the formation of actin-rich, dynamic filopodia, both along the cell periphery and the dorsal cell surface. Regulation of filopodia by LPR1 was not mediated by cdc42 or Rif, and is independent of the Arp2/3 complex. We found that LPR1 can induce filopodia formation in the absence of the Ena/Vasp family of proteins, suggesting that these molecules are not essential for the development of the protrusions. Mutagenesis experiments identified residues and regions of LPR1 that are important for the induction of filopodia. RNA interference experiments in an ovarian epithelial cancer cell line demonstrated a role for LPR1 in the maintenance of filopodia-like membrane protrusions. These observations, and our finding that LPR1 is a not an active lipid phosphatase, suggest that LPR1 may be a novel integral membrane protein link between the actin core and the surrounding lipid layer of a nascent filopodium