Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, a-kinases control actomyosin relaxation, a similar role for mammalian a-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an a-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca 2 þ -and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca 2 þ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.

SUMMARY Worldwide, acute and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knock-down in Drosophila, we report a global screen for an innate behavior and identify hundreds of novel genes implicated in heat nociception, including the α2δ-family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain evoked signals from the thalamus to higher order pain centers. Intriguingly, in α2δ3 mutant mice thermal pain and tactile stimulation triggered strong cross-activation or synesthesia of brain regions involved in vision, olfaction, and hearing.

Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized. It is shown that this FRET pair is best excited at 430 nm. We identify major sources of error and variability in confocal FRET acquisition including chromatic aberrations and instability of the excitation sources. We demonstrate that a novel correction algorithm that employs online corrective measurements yields reliable estimates of FRET efficiency, and it is also shown how the effect of other error sources can be minimized.

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The Rho family of small GTPases, including Rho, Rac, and Cdc42 isoforms, regulates different aspects of cytoskeletal organization, which are coordinated in the process of cell migration (1). Of these, Rac is involved in the protrusion of lamellipodia, which occur principally at the leading edge of migrating cells but also emerge from around newly adherent cells to mediate cell spreading (2, 3). Rac also regulates gene transcription, cell cycle progression, and transformation in vitro (4 -6) and is implicated in tumor initiation and progression in vivo (7). Rac is activated in response to various stimuli, including growth factors and adhesion to the extracellular matrix. However, how these stimuli ultimately result in Rac activation is poorly understood.The principal regulators of Rac activation are the guanine nucleotide exchange factors (GEFs) 1 and GTPase activating proteins. GEFs induce activation by exchanging GDP for GTP, whereas GTPase activating proteins enhance the intrinsic rate of hydrolysis of bound GTP to GDP, resulting in inactivation. In cells, Rac exists predominantly in its inactive GDP-bound form in a complex with RhoGDI (8). RhoGDI binds and masks the hydrophobic C-terminal region of Rac, the same region that is responsible for targeting Rac to the plasma membrane (9). Thus RhoGDI maintains Rac in the cytoplasm and must dissociate to allow Rac to translocate to the membrane and interact with membrane-associated activators (10 -12). It was shown recently (13, 14) that integrin signals disrupt the Rac-RhoGDI interaction, enabling Rac to target to regions of cell-matrix interaction and activate an adhesion-dependent signaling pathway. Thus appropriate localization, as well as activation, is necessary for Rac to carry out its functions. Increased intracellular calcium [Ca 2ϩ ] i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Previous studies (15-18) have described relationships between intracellular calcium and the activation and function of Rho family GTPases in processes including muscle contraction and the exocytic response. Intracellular calcium is also required for thrombin-and collagen-induced Rac activation in platelets (19). In neutrophils, however, chemoattractant-induced Rac activation is independent of intracellular calcium (20), suggesting that the relationship between calcium and Rac signaling is dependent on the cell type and/or the growth factor receptor involved. Ras-GRF 1 and 2, exchange factors specific for both Ras and Rac (21,22), harbor a calcium-calmodulin binding site (23) whereas the Rac exchange factor, Tiam1, is phosphorylated by calcium-calmodulin-dependent protein kinase II, which leads to increased nucleotide exchange on Rac (24). These findings suggest that nucleotide exchange on Rac may be regulated by changes in intracellular calcium. Various studies have implicated protein kinase C (PKC) in the activation of Rac. In Swiss 3T3 cells, phorbol ester treatment induces membrane ruffling, which is...