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High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds.

Contains fulltext : 70768.pdf (Publisher’s version ) (Closed access) During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

International audience; Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

Small GTPases of the Rab family are key regulators of intracellular transport. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecting these compartments. Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibitor (RabGDI). Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form. In this paper we have investigated whether Rab5, which regulates the clathrin-coated vesicle-mediated pathway of endocytosis, can directly associate with the membrane of clathrin-coated vesicles (CCV) purified from bovine brain in vitro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV. Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociation of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP. The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone. However, maximal GDP dissociation stimulation required the presence of RabGDI, suggesting that the factor(s) responsible for the membrane association and GDP/GTP exchange of Rab5 recognize the protein complexed to RabGDI. These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.

E. coli 4.5S RNA and P48 have been shown to be homologous to SRP7S RNA and SRP54, respectively. Here we report that expression of human SRP7S in E. coli can suppress the lethality caused by depletion of 4.5S RNA. In E. coli, both RNAs are associated with P48. In vitro, both E. coli P48 and SRP54 specifically bind to 4.5S RNA. Strains depleted of 4.5S RNA strongly accumulate pre-β-lactamase and fall to accumulate maltose binding protein. These effects commence well before any growth defect is observed and are suppressed by expression of human SRP7S. Strains overproducing P48 also accumulate pre-β-lactamase. 4.5S RNA and P48 are components of a ribonucleoprotein particle that we propose to be required for the secretion of some proteins.

The trafficking of intracellular membranes requires the coordination of membrane-cytoskeletal interactions. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions. We have previously identified a rho protein, rhoD, which is localized to the plasma membrane and early endosomes. When overexpressed, rhoD alters the actin cytoskeleton and plays an important role in endosome organization. We found that a rhoD mutant exerts its effect on early endosome dynamics through an inhibition in organelle motility. In these studies, the effect of rhoD on endosome dynamics was evaluated in the presence of a constitutively active, GTPase-deficient mutant of rab5, rab5Q79L. As rab5Q79L itself stimulates endosome motility, rhoD might counteract this stimulation, without itself exerting any effect in the absence of rab5 activation. We have now addressed this issue by investigating the effect of rhoD in the absence of co-expressed rab5. We find that rhoDG26V alone alters vesicular dynamics. Vesicular movement, in particular the endocytic/recycling circuit, is altered during processes such as cell motility. Due to the participation of vesicular motility and cytoskeletal rearrangements in cell movement and the involvement of rhoD in both, we have addressed the role of rhoD in this process and have found that rhoDG26V inhibits endothelial cell motility. Eur J Cell Biol

Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.

A chemically charged amber suppressor tRNA was used to introduce the photoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed the interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal sequence to be investigated during insertion into the membrane of the endoplasmic reticulum (ER). We found that different regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membrane protein) contacts the NH2-terminal region of the signal sequence while the mammalian Sec61p contacts the hydrophobic core of the signal sequence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subunits which contact distinct regions of nascent polypeptides during their membrane insertion.

SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.