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Englis

Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics

Photocatalytic Nanolithography of Self-Assembled Monolayers and Proteins

Self-assembled monolayers of alkylthiolates on gold and alkylsilanes on silicon dioxide have been patterned photocatalytically on sub-100 nm length-scales using both apertured near-field and apertureless methods. Apertured lithography was carried out by means of an argon ion laser (364 nm) coupled to cantilever-type near-field probes with a thin film of titania deposited over the aperture. Apertureless lithography was carried out with a helium–cadmium laser (325 nm) to excite titanium-coated, contact-mode atomic force microscope (AFM) probes. This latter approach is readily implementable on any commercial AFM system. Photodegradation occurred in both cases through the localized photocatalytic degradation of the monolayer. For alkanethiols, degradation of one thiol exposed the bare substrate, enabling refunctionalization of the bare gold by a second, contrasting thiol. For alkylsilanes, degradation of the adsorbate molecule provided a facile means for protein patterning. Lines were written in a protein-resistant film formed by the adsorption of oligo(ethylene glycol)-functionalized trichlorosilanes on glass, leading to the formation of sub-100 nm adhesive, aldehyde-functionalized regions. These were derivatized with aminobutylnitrilotriacetic acid, and complexed with Ni2+, enabling the binding of histidine-labeled green fluorescent protein, which yielded bright fluorescence from 70-nm-wide lines that could be imaged clearly in a confocal microscope

The Role of Mechanically Gated Ion Channels in Dorsal Closure During Drosophila Morphogenesis

<p>Physical forces play a key role in the morphogenesis of embryos. As cells and tissues change shape, grow, and migrate, they exert and respond to forces via mechanosensitive proteins and protein complexes. How the response to force is regulated is not completely understood. </p><p>Dorsal closure in Drosophila is a model system for studying cell sheet forces during morphogenesis. We demonstrate a role for mechanically gated ion channels (MGCs) in dorsal closure. Microinjection of GsMTx4 or GdCl₃, inhibitors of MGCs, blocks closure in a dose-dependent manner. UV-mediated uncaging of intracellular Ca<super>2+</super> causes cell contraction whereas the reduction of extra- and intracellular Ca<super>2+</super> slows closure. Pharmacologically blocking MGCs leads to defects in force generation via failure of actomyosin structures during closure, and impairs the ability of tissues to regulate forces in response to laser microsurgery.</p><p>We identify three genes which encode candidate MGC subunits that play a role in dorsal closure, <italic>ripped pocket</italic>, <italic>dtrpA1</italic>, and <italic>nompC</italic>. We find that knockdown of these channels either singly or in combination leads to defects in force generation and cell shapes during closure. </p><p>Our results reveal a key role for MGCs in closure, and suggest a mechanism for the coordination of force producing cell behaviors across the embryo.</p>Dissertatio

Scabrous is distributed via signaling filopodia to modulate Notch response during bristle patterning in Drosophila.

During development, cells in tissues must be patterned correctly in order to support tissue function and shape. The sensory bristles of the peripheral nervous system on the thorax of Drosophila melanogaster self-organizes from a unpatterned epithelial tissue to a regular spot pattern during pupal stages. Wild type patterning requires Notch-mediated lateral inhibition. Scabrous is a protein that can bind to and modify Notch receptor activity. Scabrous can be secreted, but it is also known to be localized to basal signaling filopodia, or cytonemes, that play a role in long-range Notch signaling. Here we show that Scabrous is primarily distributed basally, within the range of signaling filopodia extension. We show that filamentous actin dynamics are required for the distribution of Scabrous protein during sensory bristle patterning stages. We show that the Notch response of epithelial cells is sensitive to the level of Scabrous protein being expressed by the sensory bristle precursor cell. Our findings at the cell-level suggest a model for how epithelial cells engaged in lateral inhibition at a distance are sensitive local levels of Scabrous protein

Details of NsfGFP analysis in Fig 3.

12h AP pupae of the genotype NsfGFP, neur-H2B:mRFP/UAS-Scabrous-GFP; pnr-GAL4/+ were imaged for 12 hours to quantify Notch response (nuclear GFP levels). Each panel is a z-plane from one region at 2 μm steps through the tissue. (A) Region of interest from inside the pnr-GAL4 domain, which overexpresses scabrousGFP. Bristle precursor cells are labeled by neur-H2B:mRFP (red) nuclei. Example nuclear ROI for measuring NsfGFP is shown in the 4th panel to the right. Nuclear ROI measurements are taken at the z-plane where the nuclei diameter is largest. Note that at this plane, Sca-GFP puncta do not overlap with the nucleus. (B) Region of interest from outside of the pnr-GAL4 domain, in the same pupae as (A). Note the absence of scabrousGFP puncta. Example nuclear ROI for measuring NsfGFP is shown in the 3rd panel to the right. Scale bars, 5 μm. (TIF)</p

Local Scabrous expression levels are essential for robust Notch response.

(A) Example of a live pupal notum expressing UAS-Scabrous GFP in the pannier-GAL4 domain. Lateral edge of the pannier domain is marked by the dashed pink line. All Notch activated cells express the NsfGFP transcriptional reporter, which localizes GFP signal to the nucleus. Bristle precursor cells do no express NsfGFP but do express the neuralized reporter, neur-H2B:mRFP, marking their nuclei. Scale bar, 25 μm. (B) Nuclear GFP fluorescence was measured for cells adjacent to a bristle precursor cell or distant to a bristle precursor cell (+1 nuclei removed) in 14 hAP nota expressing NsfGFP. Tissues were either expressing lower amounts of Scabrous (RNAi = pannier-GAL4 > UAS-Scabrous RNAi), control levels of Scabrous (control = pannier GAL4 > UAS-white RNAi), or elevated levels of Scabrous (Sca = pannier GAL4 > UAS-Scabrous GFP). ****, pS3 Fig.</p

Actin dynamics are required for the local distribution of Scabrous.

(A) Bristle precursor cell in vivo co-expressing LifeAct-Ruby to label cell shape and ScabrousGFP. Pupae is ~14 hAP. Yellow arrowheads = Scabrous-GFP localized to signaling filopodia. Genotype: UAS-LifeAct-Ruby/UAS-scaGFP; neur-GAL4/+. Scale bar, 10 μm. (B) Timelapse panels of nota explants expressing the filamentous actin marker LifeAct-Ruby and treated with either DMSO or Cytochalasin B. Time in seconds. Either treatment was added at time = 0 seconds. Arrowheads point to individual signaling filopodia on the basal surface. Scale bars, 4 μm (CytB) and 10 μm (DMSO). Genotype: UAS-LifeAct-Ruby/+; pnr-GAL4/+. (C) Immunofluorescence image of a sensory bristle precursor cell from a nota under control conditions for 30 minutes prior to fixation. Signaling filopodia are still visible in F-actin panel. White arrow in Scabrous panel points to intracellular Scabrous. Yellow arrow in Scabrous panel points to local Scabrous. Scale bar, 10 μm. Genotype: neur-GAL4, UAS-GMCA/+. (D) Examples cell morphologies upon DMSO control or Cytochalasin B treatments for 5 minutes. Green, anti-GFP (GFP-actin binding domain of moesin labeling F-actin). Red, anti-Scabrous. Scale bar, 5 μm. Genotype: neur-GAL4, UAS-GMCA/+. (E) Quantification of signaling filopodia length after 5 minutes of treatment with either DMSO (n = 35 filopodia across 17 cells) or Cytochalasin B (n = 36 filopodia across 29 cells). ****, pS2 Fig.</p

Volume rendering of apicobasal distribution of Scabrous protein in a bristle precursor cell.

Green = anti-GFP. Red = anti-Scabrous. Genotype: neur-GAL4, UAS-GMCA/+. Scale bars in microns. Apical plane at (0,0,0). (AVI)</p