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Abstract: Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in children and young adults. NPHP8/RPGRIP1L is a novel ciliary gene that, when mutated, in addition to causing NPHP, also causes Joubert syndrome (JBTS) and Meckel syndrome (MKS). The exact function of NPHP8 and how defects in NPHP8 lead to human diseases are poorly understood. Here, we studied the Caenorhabditis elegans homolog nphp-8 (C09G5.8) and explored the possible function of NPHP-8 in ciliated sensory neurons. We determined the gene structure of nphp-8 through rapid amplification of cDNA ends (RACE) analysis and discovered an X-box motif that had been previously overlooked. Moreover, NPHP-8 co-localized with NPHP-4 at the transition zone at the base of cilia. Mutation of nphp-8 led to abnormal dye filling (Dyf) and shorter cilia lengths in a subset of ciliary neurons. In addition, chemotaxis to several volatile attractants was significantly impaired in nphp-8 mutants. Our data suggest that NPHP-8/RPGRIP1L plays an important role in cilia formation and cilia-mediated chemosensation in a cell type-specific manner. [Copyright &y& Elsevier]

Abstract: Previously, we identified a clathrin-dependent slow endocytosis and a clathrin-independent fast endocytosis in pancreatic β cells, both triggered by elevated cytoplasmic Ca2+ concentration. In the current study, we attempted to explore the roles of different dynamin isoforms in these endocytotic processes. We first confirmed the existence of both neuron-specific dynamin 1 and ubiquitous dynamin 2 in INS-1 cells using both quantitative RT-PCR and Western blot experiments. By specifically knocking down the endogenous level of either dynamin isoform from INS-1 cells, we showed that dynamin 1 and dynamin 2 simultaneously participate in the clathrin-independent and -dependent membrane retrieval in pancreatic β cells. Transferrin internalization was also inhibited in cells with knock down of both dynamin 1 and dynamin 2. Based on these results, we argue that different dynamin isoforms play overlapping roles in different types of endocytosis. [Copyright &y& Elsevier]

Abstract: Membrane syntaxin plays essential roles in exocytosis in eukaryotic cells. The conservative Habc domain in plasma membrane syntaxins implies important roles for syntaxin targeting and function. Our previous study showed Habc domain was necessary for the trafficking and cluster distribution of syntaxin 1A on the plasma membrane. Here we identified which of the three domains (Ha, Hb and Hc) was essential for Stx1A trafficking and clustering. We found that, in INS-1 cells, the mutant truncated with either Ha, Hb or Hc domain could be sorted to the cell surface by a different mechanism compared to that of whole Habc truncated mutant. In contrast to wild type Stx1A, none of the mutants showed cluster distribution at the functional sites, suggesting that the physiological localization of Stx1A relies on intact Habc domain. Furthermore Munc18-1 is found not to be essential for Stx1A cluster distribution, despite important role in stabilizing membrane delivery of Stx1A. [Copyright &y& Elsevier]

Abstract: STIM1 and Orai1 have recently been identified to be crucial in the regulation of store-operated Ca2+ entry. However, it remains to be established how STIM1 couples store depletion to the functioning of Orai1 in the plasma membrane. Using quantitative measurement, we find little STIM1 on the surface membrane which is not increased by store depletion. We further demonstrate that Orai1 assembles into clusters that co-localize with STIM1 aggregations upon store depletion. The clustering of Orai1 is only seen when Oari1 are co-expressed with STIM1, but not when expressed alone. Moreover, ER retreat from cell periphery leads to mismatching of Orai1 and STIM1 puncta. Therefore, we propose that store depletion causes aggregation and translocation of STIM1 in close apposition to the plasma membrane, which in turn recruits Orai1 in the plasma membrane to the sites of STIM1 aggregates to assemble functional units of CRAC channels in a stoichiometric manner. [Copyright &y& Elsevier]