The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis

Acknowledgements We wish to thank Jorge Galán, Gregory Pazour, Derek Toomre, Giuliano Callaini, Joel Rosenbaum, Alessandra Boletta and Francesco Blasi for generously providing reagents and for productive discussions, and Sonia Grassini for technical assistance. The work was carried out with the financial support of Telethon (GGP11021) and AIRC.Peer reviewedPostprin

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN

Tumor Susceptibility Gene 101 (TSG101) Is a Novel Binding-Partner for the Class II Rab11-FIPs

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway

Trafficking of Sendai Virus Nucleocapsids Is Mediated by Intracellular Vesicles

Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.To analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Collectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3

Rab11-FIP2 influences multiple components of the endosomal system in polarized MDCK cells

The Rab11 Family Interacting Proteins (Rab11-FIPs) are hypothesized to regulate sequential steps in the apical recycling and transcytotic pathways of polarized epithelial cells. Previous studies have suggested that Rab11-FIP proteins assemble into multi-protein complexes regulating plasma membrane recycling. Rab11-FIP2 interacts with both myosin Vb and Rab11. Recent investigations have noted that that Rab11-FIP2 mutants [Rab11-FIP2(129–512), also designated Rab11-FIP2(ΔC2) and Rab11-FIP2(S229A, R413G), also designated Rab11-FIP2(SARG)], are potent inhibitors of transcytosis in polarized MDCK cells. Interestingly, Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), also altered the morphology of the EEA-1 positive early endosomal compartment. These findings suggested that Rab11-FIP2 mutants could differentiate different points along the recycling pathway. We therefore sought to investigate whether Rab11-FIP2 is a general regulator of the early endosomal system. Both Rab11-FIP2 mutants altered the localization and co-localized with dynein heavy chain. In contrast, both clathrin heavy chain and AP-1 accumulated with membranes containing Rab11-FIP2(SARG), but not with Rab11-FIP2(ΔC2). Expression of Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), caused clustering of early endosomal markers Rab5b, Epsin 4 and IQGAP1, around a collapsed Rab11-FIP2 containing membranous cisternum. Interestingly, neither Rab11-FIP2 mutant had any effect on the distribution of Rab5a, a classical early endosome marker. The results support the view that Rab11-FIP2 may influence microtubule-dependent centripetal movement of subsets of early endosomes as well as processing through the common and recycling endosomal systems