Aurora A-dependent CENP-A phosphorylation at inner centromeres protects bioriented chromosomes against cohesion fatigue.

Sustained spindle tension applied to sister centromeres during mitosis eventually leads to uncoordinated loss of sister chromatid cohesion, a phenomenon known as "cohesion fatigue." We report that Aurora A-dependent phosphorylation of serine 7 of the centromere histone variant CENP-A (p-CENP-AS7) protects bioriented chromosomes against cohesion fatigue. Expression of a non-phosphorylatable version of CENP-A (CENP-AS7A) weakens sister chromatid cohesion only when sister centromeres are under tension, providing the first evidence of a regulated mechanism involved in protection against passive cohesion loss. Consistent with this observation, p-CENP-AS7 is detected at the inner centromere where it forms a discrete domain. The depletion or inhibition of Aurora A phenocopies the expression of CENP-AS7A and we show that Aurora A is recruited to centromeres in a Bub1-dependent manner. We propose that Aurora A-dependent phosphorylation of CENP-A at the inner centromere protects chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules.

EB1 interacts with outwardly curved and straight regions of the microtubule lattice.

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

Rab24 is required for normal cell division.

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.

Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes.

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells.

Structural basis for recruitment of Rab6-interacting protein 1 to Golgi via a RUN domain.

Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 angstroms resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment.

Rab6-interacting protein 1 links Rab6 and Rab11 function.

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.

Rab11A controls the biogenesis of Birbeck granules by regulating Langerin recycling and stability.

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

Purification and properties of rab6 interacting proteins.

A crucial step in the characterization of novel partners of Rab proteins is the confirmation that they indeed interact together by techniques other than the yeast two-hybrid assay used to discover them. Some methods and clues that would help to discriminate between putative interactors are summarized. Pull-down, co-immunoprecipitation, and gel filtration experiments are described as ways of checking protein-protein interaction in vitro and in vivo.

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression.

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

Recombinant antibodies to the small GTPase Rab6 as conformation sensors.

Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.

Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport.

Rab6 GTPase regulates intracellular transport at the level of the Golgi complex. Using the yeast two-hybrid screen, we have isolated two clones that specifically interact with the three isoforms of Rab6 present in mammalian cells (Rab6A, A' and B). The cDNAs encode two proteins of 976 and 1120 amino acids (calculated molecular mass of 112 and 128 kDa, respectively) that we named Rab6IP2A and Rab6IP2B (for Rab6 Interacting Protein 2). The two proteins likely correspond to spliced variants of the same gene. Rab6IP2s have no significant homology with other known proteins, including Rab effectors or partners. They are ubiquitously expressed, mostly cytosolic and found in high molecular mass complexes in brain cytosol. We show that Rab6IP2s can be recruited on Golgi membranes in a Rab6:GTP-dependent manner. The overexpression of any form of Rab6IP2 has no detectable effect on the secretory pathway. In contrast, the retrograde transport of the Shiga toxin B subunit between the plasma membrane and the Golgi complex is partly inhibited in cells overexpressing the Rab6-binding domain of Rab6IP2. Our data suggest that Rab6IP2s is involved in the pathway regulated by Rab6A'.