Rgnef (p190RhoGEF) Knockout Inhibits RhoA Activity, Focal Adhesion Establishment, and Cell Motility Downstream of Integrins

Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions. Rho-family GTPases are molecular switches that regulate actin and focal adhesion dynamics in cells. Guanine nucleotide exchange factors (GEFs) activate Rho-family GTPases. Rgnef (p190RhoGEF) is a ubiquitous 190 kDa GEF implicated in the control of colon carcinoma and fibroblast cell motility.Rgnef exon 24 floxed mice (Rgnef(flox)) were created and crossed with cytomegalovirus (CMV)-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development. Heterozygous Rgnef(WT/flox) (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, but Rgnef(flox/flox) (Cre+) mice numbers at 3 weeks of age were significantly less than expected. Rgnef(flox/flox) (Cre+) (Rgnef-/-) embryos and primary mouse embryo fibroblasts (MEFs) were isolated and verified to lack Rgnef protein expression. When compared to wildtype (WT) littermate MEFs, loss of Rgnef significantly inhibited haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. In WT MEFs, Rgnef activation occurs within 60 minutes upon fibronectin plating of cells associated with RhoA activation. Rgnef-/- MEF phenotypes were rescued by epitope-tagged Rgnef re-expression.Rgnef-/- MEF phenotypes were due to Rgnef loss and support an essential role for Rgnef in RhoA regulation downstream of integrins in control of cell migration

The Minimal Autoinhibited Unit of the Guanine Nucleotide Exchange Factor Intersectin

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain

Magnetic properties experiments and the Surface Stereo Imager calibration target onboard the Mars Phoenix 2007 Lander: Design, calibration, and science goals

The first NASA scout mission to Mars, Phoenix, launched 4 August will land in the northern part of Mars in the locality of 68 degrees N and 233 degrees E on 25 May 2008. Part of the science payload is the Magnetic Properties Experiments (MPE) that consists of two main experiments: the Improved Sweep Magnet Experiment (ISWEEP) and 10 sets of two Microscopy, Electrochemistry, and Conductivity Analyzer ( MECA) magnet substrates with embedded permanent magnets of different strength. The ISWEEP experiment is, as the name indicates, an improved version of the Sweep Magnet Experiments flown onboard the two Mars Exploration Rovers (MERs) Spirit and Opportunity. The sweep magnet is ring shaped and is designed to allow only nonmagnetic particles to enter a small circular area at the center of the surface of this structure. Results from this experiment have shown that on the MERs hardly any particles can be detected in the central area of this ring-shaped magnet. From this we have concluded that essentially all particles in the Martian atmosphere are magnetic in the sense that they are attracted to permanent magnets. In order to improve the sensitivity of the Sweep Magnet Experiment for detection of nonmagnetic or very weakly magnetic particles, the ISWEEP holds six ring-shaped magnets, somewhat larger than the sweep magnet of the MERs, and with six different background colors in the central area. The six different colors provide new possibilities for improved contrast between these background colors, i.e., any putative nonmagnetic particles should render these more easily detectable. The Surface Stereo Imager will also take advantage of the small clean areas in the ISWEEPs and use the presumably constant colors for radiometric calibration of images. The MECA magnets work as substrates in the MECA microscopy experiments; they are built to attract and hold magnetic particles from dust samples. The collected dust will then be examined by the optical microscope and the atomic force microscope in the MECA package

Hippocampal long-term potentiation and neural cell adhesion molecules L1 and NCAM

Synaptic membranes express cell adhesion molecules. Here we investigate the role of the neural cell adhesion molecules L1 and NCAM in hippocampal long-term potentiation (LTP), a sustained-use-dependent increase in synaptic efficacy that has been implicated in learning and memory. L1 and NCAM mediate cell interactions during neural development and are strongly expressed in the hippocampus. They cooperate to strengthen L1-dependent cell adhesion and are coupled to second messenger pathways. We show that LTP in CA1 neurons of rat hippocampal slices was reduced by application of various L1 and NCAM antibodies, recombinant L1 fragments, and upon dissociation of the L1/NCAM complex through oligomannosidic carbohydrates and NCAM peptides. Neither the activation of NMDA (N-methyl-D-aspartate) receptors nor the maintenance of LTP was affected. These results suggest that L1 and NCAM modulate the development or the stabilization of LTP