A kinetoplastid‐specific kinesin is required for cytokinesis and for maintenance of cell morphology in Trypanosoma brucei

Abstract

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.

An orphan kinesin in trypanosomes cooperates with a kinetoplastid-specific kinesin to maintain cell morphology through regulating subpellicular microtubules

“…RNAi of TbKIN-C caused similar phenotype (Hu et al, 2012). It remains unknown how the new flagellum and the small portion of cytoplasm were split from the main cell body after TbKIN-D or TbKIN-C RNAi.…”

“…We find that RNAi of TbKIN-D resulted in distorted cell morphology and disorganization of subpellicular microtubules. We also demonstrate that TbKIN-C, a trypanosomespecific kinesin previously shown to be essential for maintaining cell morphology in T. brucei (Hu et al, 2012), associates with TbKIN-D in vivo. Our data suggest a cooperative role of the two kinesin-like proteins in cell morphogenesis through regulating subpellicular microtubules.…”

“…7B). Liquid chromatography-tandem mass spectrometry (LC/ MS/MS) analysis indicated that this protein was TbKIN-C, a kinetoplastid-specific kinesin (Hu et al, 2012). To further confirm the in vivo interaction between TbKIN-D and TbKIN-C, coimmunoprecipitation was performed using cells co-expressing endogenously PTP-tagged TbKIN-D and triple HA-tagged TbKIN-C. We found that immunoprecipitation of TbKIN-D-PTP was able to pull down TbKIN-C-3HA from the cell lysate (Fig.…”

“…Since TbKIN-D and TbKIN-C contain three coiledcoil motifs at their C-termini (Fig. 1A) (Hu et al, 2012), we tested whether the coiled-coil motif is required for interaction between them. Yeast two-hybrid assays indicated that TbKIN-D and TbKIN-C were capable of interacting with each other, but neither protein was able to interact with itself (Fig.…”

“…RNAi of one orphan kinesin (Tb927.11.10760) resulted in severe growth inhibition (see below). We named this kinesin TbKIN-D after the three kinesins, TbKIN-A, TbKIN-B and TbKIN-C, identified in our previous studies (Li et al, 2008b;Hu et al, 2012). TbKIN-D possesses an N-terminal motor domain and three coiled-coil motifs in the C-terminus (Fig.…”