Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P 2 . Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P 2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.Small guanosine triphosphatases (GTPases) from the Ras, Rho, Arf, and Rab subfamilies often exert their role at the PM where they control diverse signaling, cytoskeletal, and transport processes (1-3). KRas, CDC42, and other family members require a cluster of positively charged amino acids for PM localization and activity (2, 4). In vitro studies indicate that the physiological PM binding partner of such polybasic clusters could be phosphatidylserine, which has one negative charge, or the less abundant lipid second messenger PI(4,5)P 2 , which has four negative charges (5-7). We took a genomic survey approach and investigated PM-targeting mechanisms by confocal imaging of 125 cyan fluorescent protein (CFP)-tagged constitutively active small GTPases (8). Expression in NIH3T3 and HeLa cells showed that 48 small GTPases were fully or partially localized to the PM (Fig. 1A and fig. S1).Thirty-seven of these PM-localized small GTPases had C-terminal polybasic clusters consisting of four or more Lys or Arg residues at positions 5 to 20 from the C terminus ( Fig. 1B and fig. S1). Polybasic clusters were found in three forms: They were present together with N-terminal myristoylation consensus sequences (as in Arl4) (9) or with C-terminal prenylation consensus sequences (as in KRas) (5, 6, 10), or they lacked lipid modifications (as in Rit) (11). We called these three combinations polybasic-myristoyl, polybasic-prenyl, and polybasic-nonlipid PM-targeting motifs, respectively. A number of remaining PMtargeted small GTPases had a combined prenylation and palmitoylation consensus sequence that mediated PM targeting without requiring polybasic amino acids (as does that of HRas) (Fig. 1D).To test whether polybasic clusters are anchored to the PM by binding to PI(4,5)P 2 (14), we hydrolyzed PM PI(4,5)P 2 by rapid targeting of Inp54p, a 5′ specific PI(4,5)P 2 phosphatase (15), to the PM. This method is based on a PM-localized FK506-binding protein (FKBP12)-rapamycin-binding (FRB) construct and a cytosolic Inp54p enzyme conjugated with FKBP12 (CF-Inp) that can be translocated to the PM by chemical heterodimerization by using a rapamycin analog, iRap (16).In experiments where we monitored PI(4,5)P...

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase-mediated signaling processes.The Rho family of small GTPases has been shown to function as molecular switches in several physiological contexts such as cell migration, phagocytosis, adhesion and secretion, as well as transcriptional control 1,2 . Rho GTPase signaling processes are known to have considerable crosstalk 3 , and they regulate cellular events in a concerted and integrated manner that is only partially understood. Previous studies addressing specific roles of small GTPases often relied on the expression of constitutively active and inactive mutated forms of Rho, Cdc42 and Rac. But these GTPases have limited use because they are active for time periods that exceed the duration of the signaling process they control so that transient activities cannot be investigated. Thus, rapid conditional perturbation would be more useful for time-resolved studies of these signaling proteins.A conceptually elegant approach for Rac and Cdc42 activation that takes advantage of rapamycin-induced heterodimerization has been reported [4][5][6] . In this study, researchers conjugated Rho GTPase proteins with a rapamycin-binding domain of mTOR (FRB) and the cytoplasmic region of a transmembrane receptor was conjugated with a FK506-binding protein (FKBP). Their goal was to activate downstream processes by using a small molecule-dependent translocation of mutant Rho GTPases to the plasma membrane. Rac or Cdc42 membrane recruitment in this system, however, was not sufficient to trigger phagocytosis or membrane extension and substantial physiological responses were only induced after clustering of many receptors using antibody-coated beads. We have developed a more rapid and direct perturbation method for Rho GTPases by optimizing the inducible plasma membrane targeting, synthesizing a rapamycin analog that can trigger rapid plasma membrane translocation and engineering different enzymatic fusion constructs.To evaluate how to best target Rho GTPases to the plasma membrane, we tested a Lyn Nterminal sequence not previously used for heterodimerization (Supplementary Methods

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

We present a versatile platform to inactivate proteins in living cells using light, light-activated reversible inhibition by assembled trap (LARIAT), which sequesters target proteins into complexes formed by multimeric proteins and a blue light-mediated heterodimerization module. Using LARIAT, we inhibited diverse proteins that modulate cytoskeleton, lipid signaling and cell cycle with high spatiotemporal resolution. Use of single-domain antibodies extends the method to target proteins containing specific epitopes, including GFP.

The Ca 2؉ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

Primary cilia exert a profound impact on cell signalling and cell cycle progression. Recently, actin cytoskeleton destabilization has been recognized as a dominant inducer of ciliogenesis, but the exact mechanisms regulating ciliogenesis remain poorly understood. Here we show that the actin cytoskeleton remodelling controls ciliogenesis by regulating transcriptional coactivator YAP/TAZ as well as ciliary vesicle trafficking. Cytoplasmic retention of YAP/TAZ correlates with active ciliogenesis either in spatially confined cells or in cells treated with an actin filament destabilizer. Moreover, knockdown of YAP/TAZ is sufficient to induce ciliogenesis, whereas YAP/TAZ hyperactivation suppresses serum starvation-mediated ciliogenesis. We also identify actin remodelling factors LIMK2 and TESK1 as key players in the ciliogenesis control network in which YAP/TAZ and directional vesicle trafficking are integral components. Our work provides new insights for understanding the link between actin dynamics and ciliogenesis.