Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome.

Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

A Combination of Screening and Computational Approaches for the Identification of Novel Compounds That Decrease Mast Cell Degranulation.

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.

Survival of the weakest: signaling aided by endosomes.

The tyrosine kinase receptor c-Met plays a key role in cell proliferation, morphogenesis, and motility in response to hepatocyte growth factor. C-Met is often altered in cancer and is a major target for therapeutic intervention. Despite knowing a great deal of the molecular machinery downstream of this receptor tyrosine kinase, the spatiotemporal regulation of c-Met signaling still remains elusive. In this issue of the Journal of Cell Biology, Kermorgant and Parker (Kermorgant, S. and P.J. Parker. 2008. J. Cell Biol. 182:855-863) provide evidence for a model in which the c-Met-activated STAT3 signal is mediated by endosomal trafficking. This study elegantly highlights how weak signals can be effectively transmitted to the nucleus by exploiting endosomal compartments, raising important mechanistic implications for the signaling research community.

Analysis of fusion using a virus-free cell fusion assay.

For enveloped viruses, such as viruses within the herpesvirus family, of which Epstein-Barr virus (EBV) is a member, infection of target cells includes two distinct steps. The first is characterized by the binding of viral envelope glycoproteins to host cellular receptors. After binding, the viral membrane and the cellular membrane fuse. Without both binding and fusion, the virus is not able to enter the host target cell efficiently. Combined with the specific tropism of EBV for primarily two cell types, B lymphocytes and epithelial cells, and the difficulty in inducing lytic replication of EBV in vitro, there is a lack of a good experimental model to study EBV-induced viral fusion. To study fusion more efficiently and effectively, we have employed a virus-free cell-cell fusion assay. In the effector cell, the viral glycoproteins and a plasmid containing the T7 promoter, driving the luciferase gene, are expressed. In the target cell type, T7 RNA polymerase is transfected. Fusion is quantitated by the amount of luciferase expression after mixing of the two cell types. Alongside the fusion assay, a CELISA is performed to determine glycoprotein expression on the effector cells. This methodology has been useful in studying membrane fusion induced by other herpesvirus family members.

Cell-surface expression of a mutated Epstein-Barr virus glycoprotein B allows fusion independent of other viral proteins.

Epstein-Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lines (AGS and SCC68) or the human embryonic kidney cell line 293-P. Fusion with human embryonic kidney 293-P cells was greater than fusion observed with B cells, indicative of an important role for cell contact. An antibody directed against the gH and gL complex inhibited epithelial cell fusion. Increased surface expression of gB alone as a result of truncations or point mutants in the carboxyl-terminal tail allowed gB-mediated fusion with epithelial cells, albeit at a lower level than with coexpression of gB, gH, and gL. Overall, gB appears to be the critical component for EBV glycoprotein-mediated cell fusion.

Mutational analysis of the HLA class II interaction with Epstein-Barr virus glycoprotein 42.

Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.