Kinesins are motor-based transport proteins that play diverse roles in various cellular processes. The trypanosome genome lacks the homologs of many conserved mitotic kinesins, but encodes a number of trypanosome-specific kinesins with unknown function. Here, we report the biochemical and functional characterization of TbKIN-C, a trypanosome-specific kinesin, which was initially identified through an RNAi screen for cytokinesis genes in T. brucei. TbKIN-C possesses in vitro ATPase activity and associates with cytoskeletal tubulin microtubules in vivo. It is distributed throughout the cytoskeleton with a focal enrichment at the posterior end of the cell during early cell cycle stages. RNAi of TbKIN-C resulted in distorted cell shape with an elongated posterior filled with tyrosinated tubulin microtubules. Silencing of TbKIN-C impaired the segregation of organelles and cytoskeletal structures and led to detachment of the new flagellum and a small portion of the cytoplasm. We also show that RNAi of TbKIN-C compromised cytokinesis and abolished the trans-localization of TbCPC1, a subunit of the chromosomal passenger complex, from the central spindle to the initiation site of cytokinesis. Our results suggest an essential role of TbKIN-C in maintaining cell morphology, likely through regulating microtubule dynamics at the posterior end of the cell.

SummaryMicrotubules are a vital part of the cytoskeleton of eukaryotic cells and are involved in various cellular processes. The cytoskeleton of Trypanosoma brucei is characterized by an array of subpellicular microtubules and is essential for maintenance of cell shape and polarity, but little is known about the regulation of the assembly and organization of the subpellicular microtubule corset. Here, we report that the orphan kinesin TbKIN-D regulates the organization of subpellicular microtubules and is required for maintaining cell morphology. TbKIN-D possesses in vitro ATPase activity, associates with cytoskeletal microtubules and is distributed throughout the cytoskeleton at all cell cycle stages. RNAi of TbKIN-D disrupts the organization of the subpellicular microtubule corset and distorts cell morphology, resulting in round cells with an elongated posterior filled with newly assembled microtubules. Depletion of TbKIN-D also abolishes the segregation of organelles and cytoskeletal structures, suggesting that cellular morphogenesis is essential for proper organelle segregation. Moreover, TbKIN-D deficiency impairs the attachment of the new flagellum without compromising the formation of the flagellum attachment zone. Finally, we identified TbKIN-C, a kinetoplastid-specific kinesin known to regulate subpellicular microtubules and cell morphogenesis in T. brucei, as a partner of TbKIN-D. Further, we demonstrate that interaction between TbKIN-C and TbKIN-D requires the coiled-coil motifs in the C-termini of both proteins. Altogether, our results suggest that TbKIN-D cooperates with TbKIN-C to maintain cell morphology by regulating the organization of the subpellicular microtubule corset.

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Induction of Ag-specific regulatory T cells (iTregs) by vaccination is a promising strategy for treating autoimmune diseases. We previously demonstrated that DNA and protein covaccination converted naive T cells to Ag-specific iTregs by inducing CD11c+CD40lowIL-10+ regulatory dendritic cells (DCregs). However, it is unclear how coimmunization induces the DCregs. In this paper, we report that the event is initiated by coentry of sequence-matched DNA and protein immunogens into the same DC via caveolae-mediated endocytosis, which leads to inhibition of phosphorylation of caveolin-1 (Cav-1), the main component of caveolae, and upregulation of Tollip. This triggers downstream signaling that upregulates suppressor of cytokine signaling 1 and downregulates NF-κB and STAT-1α. Silencing either Cav-1 or Tollip blocks the negative signaling, leading to upregulated expression of CD40, downregulated production of IL-10, and loss of iTreg-inducing function. We further show that DCregs can be induced in culture from primary DCs and JAWS II DC lines by feeding them sequence-matched DNA and protein immunogens. The in vitro-generated DCregs are effective in ameliorating autoimmune and inflammatory diseases in several mouse models. Our study thus suggests that DNA and protein coimmunization induces DCregs through Cav-1– and Tollip-mediated negative signaling. It also describes a novel method for generating therapeutic DCregs in vitro.

Background: MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs, which usually bind to the 3'-untranslated region of target mRNAs and are capable of inducing posttranscriptional gene regulation by blocking translation or by degrading the target mRNA. However, the expression level of miR-23 in radiation induced carcinogenesis is largely unknown. Methods: Radiation induced thymic lymphoma model in BALB/c mice was set up. miR-23a & miR-23b miRNA levels in different tissues and cells were detected by real-time qPCR. miR-23a/b inhibitor and miR-23a/b mimics were transfected to lymphoma cells and the target of miR-23a/b was identified by microRNA target prediction and Luciferase assays. Results: We found that miR-23a & miR-23b were up-regulated in radiation induced thymic lymphoma tissue samples. Cell death and apoptosis were increased by miR-23a/b inhibitor and decreased by miR-23a/b mimics in lymphoma cells. Computational analysis found a putative target site of miR-23a/b in the 3′UTR of Fas mRNA, which was verified by luciferase reporter assay. Forced over-expression of miR-23a/b decreased the level of Fas protein. Moreover, over-expression of Fas rescued the pro-proliferation effect of miR-23, indicating Fas is a direct mediator of miR-23 functions. Furthermore, contrast to miR-23a/b which was up regulated, the Fas expression level was down-regulated and inversely correlated with miR-23 in split radiation induced lymphoma tissue samples. Finally, our data also indicates that miR-23a could repress Fas much more potent than miR-23b and the additional region besides conserved seed pairing enables miR-23a's higher regulation. Conclusions: In this study, using a radiation induced thymic lymphoma model in BALB/c mice, We conclude that the expression of miR-23a/b is up-regulated in radiation-induced thymic lymphoma and it maybe a novel therapeutic target of that cancer.