Most vertebrate cells are ciliated and the genesis and resorption of these cilia are coordinated with progression through the cell cycle. However, no direct causal relationship between the two processes has been demonstrated. The assembly and maintenance of cilia are mediated by intraflagellar transport (IFT), a process by which multimeric protein complexes, called IFT particles, move bidirectionally along the cilium to shuttle axonemal precursors to the distal tip for assembly and turnover products back to the cell body for removal. Recently, RNAi knockdown of a core IFT particle protein, IFT27, resulted in cell-cycle defects in addition to defects of flagellar assembly in the green alga, Chlamydomonas reinhardtii. Because sequence analysis of IFT27 reveals significant similarity to proteins of the Rab family of guanosine triphosphatases (GTPases), it is hypothesized that IFT27 may be functioning as a regulator of the cell cycle in a way similar to that observed for Rabs in higher organisms. To investigate this hypothesis, the cellular localization of IFT27 will be examined by immunofluorescence microscopy in order to determine if the distribution of IFT27 changes in concert with the cell cycle. Next, an IFT27-containing sucrose gradient fraction will be obtained from cytoplasmic protein extracts prepared during the cell cycle stage at which IFT27 is observed to localize most prominently to the cell body interior. To identify proteins that bind to IFT27 during the cell cycle, the cytoplasmic IFT27-containing fraction will be subjected to immunoprecipitation with IFT27 antibodies and the resulting coimmunoprecipitates will be identified by gel electrophoresis and mass spectrometry. To assess the function of these cytoplasmic binding partners of IFT27, each will be knocked down by RNAi. Knockdowns will be examined for defects in cell cycle progression, cell division and flagellar assembly. The relevance of these studies to human health is supported by the rapidly emerging body of evidence linking defects in the assembly and maintenance of cilia to a growing list of human diseases including Bardet-Biedl syndrome, and polycysitic kidney disease (PKD). The fact that a decrease in IFT27 causes loss of the cilium, taken together with the fact that the presence of IFT27 is required for completion of mitosis and cytokinesis provides a possible molecular link between the cilium and diseases of cell proliferation. ? ? ?
|Wood, Christopher R; Wang, Zhaohui; Diener, Dennis et al. (2012) IFT proteins accumulate during cell division and localize to the cleavage furrow in Chlamydomonas. PLoS One 7:e30729|