The Blogging of Webster 582

An alternative mechanism is based on the idea that Rac, Rho, and Cdc42 each have many effectors and some effector pathways inhibit migration and invasion . Interestingly, our previous work also implicated GDI1 in the activation mechanism for Rac . Thus, it seems possible that GDI2 might direct activity of a Rho GTPase toward a specific pathway that inhibits metastasis. Given that GDI2 has the highest affinity for Rac1, this GTPase is the main candidate. However, the general importance of Rac1 in metastasis precludes testing this idea using the experimental metastasis model by globally inhibiting Rac1. But as a first step, we were able to assess effects of RhoGDI constructs on Rac1 activity.
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Here, we observed that deletion of either RDI1 or of one of the Cdc42 GAPs results in suppression of the mitotic exit defect of Δlte1 cells. Consistently, overexpression of either STE20, GIC1, or GIC2 has the same effect on cells deleted for LTE1 (Höfken and Schiebel, 2002; Höfken and Schiebel, 2004). This suggests that deletion of the GAP genes or RDI1 leads to a slight activation of Cdc42 and its effectors, which trigger exit from mitosis independently of Lte1 .
Albicans shows a similar defect in filamentation, when grown on solid medium . For agar invasion assays, 105 cells of an overnight culture were spotted on YPD and grown for 2 d at 30°C. Plates were photographed before and after being rinsed under a gentle stream of deionized water. For pseudohyphal growth assays, cells were grown overnight, and 100 cells were spread on solid SLAD medium. Plates were incubated for 4 d at 30°C, and images were taken using a 10× objective.
We next investigated the mechanism by which RhoG suppression is reversed by FGF2/S4 signaling. Previous studies indicate that S4 oligomerization leads to the activation of PKCα (Oh et al., 1997; Horowitz et al., 2002). To explore the role of this kinase in RhoG activation, we examined the effect of constitutively active PKCα on baseline RhoG activity. RFPECs transfected with myristoylated PKCα showed increased RhoG activation at baseline (Fig. 4 a). Conversely, a dominant-negative PKCα construct blocked FGF2-induced RhoG activation (Fig. S1 i).

Consistently, in Δcla4 cells a slightly higher portion of Rho1 was found in the cytosol compared with the wild-type strain, whereas the cytosolic amount of Rho1 in the Δcla4 Δrdi1 double mutant was similar to the wild-type and Δrdi1 . For Cdc42, there was only a weak or no effect for Cdc42 in the CLA4 deletion strain . Finally, using the membrane extraction assay it was tested whether Cla4 antagonizes Rdi1. Whereas higher Rdi1 levels increased the cytosolic pool of Cdc42 and Rho1 , simultaneous overexpression of CLA4 and RDI1 reversed this effect . Under these conditions, cytosolic amounts of Cdc42 and Rho1 are comparable with the wild-type situation.
By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

The quantitative analysis of translocation was performed using density analysis software from FluorChem® Imaging Systems . Images were taken using Alpha Innotech FluorChem™ 8000 imaging system for densitometry analysis with AlphaChem™ imaging software . Cerevisiae, we analyzed the phenotype of cells deleted for and overexpressing RDI1, respectively. The RDI1 deletion strain was indistinguishable from wild-type cells in terms of growth, cell morphology, and mating . Albicans loss of the homologous gene RDI1 greatly reduces filamentous growth .
Cardiac organogenesis is characterized by the precise temporal and region-specific regulation of cell proliferation, migration, death, and differentiation (reviewed by Sucov, 1998; Fishman and Chien, 1997). In mouse embryos, cardiac mesoderm involutes during gastrulation and becomes specified when it reaches its position bilaterally in the anterior lateral plate mesoderm by 7.0 days post coitus (E7.0). As neurulation proceeds, the bilateral precardiac cells then migrate toward the midline and fuse to form the definitive heart tube by E8.0.
Their moderate differences in extraction kinetics might nonetheless be biologically meaningful. Cy3-Rho and Cy3-Cdc42, bound to GDI for stabilization, were microinjected into oocytes 41% and 53% above endogenous levels, respectively. Both Cy3-Rho and Cy3-Cdc42 localized to wounds , in a manner indistinguishable from their IT counterparts expressed in the oocyte . As observed with IT-Rho and IT-Cdc42, Cy3-Rho completely overlapped with the zone of Rho activity while Cy3-Cdc42 localized throughout and slightly interior to the active Cdc42 zone. These results indicate that the IT- and Cy3-tagged GTPase variants faithfully mimic endogenous GTPases during cell wound repair. The Rho family GTPases, including Rho, Rac and Cdc42, are essential signaling proteins that mediate morphological changes in cells by directing local cytoskeletal rearrangements (Bishop and Hall, 2000; Kimura et al., 1996).

As a further means to avoid "overselling", we removed the clause "While these findings might seem heretical" from the Discussion paragraph that now begins "This finding has the virtue…". Phosphorylation at Ser96 in RhoGDI1 is required for CCK-induced RhoA and Rac1 activation. Pancreatic acini overexpressing mutant S96D-RhoGDI1 exhibit a RhoA and Rac1-binding deficiency. Schematic of RhoGDI’s role in RhoGTPase zone definition around wounds.