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

Abstract

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.

The Cooperative Roles of Two Kinetoplastid-Specific Kinesins in Cytokinesis and in Maintaining Cell Morphology in Bloodstream Trypanosomes

“…We previously identified two trypanosome-specific orphan kinesins, TbKIN-C and TbKIN-D, that cooperate to maintain cell morphology by regulating the organization of the subpellicular microtubule corset in the procyclic form [15], [16]. Both kinesins associate with the microtubule cytoskeleton, but TbKIN-C is additionally enriched at the posterior tip of the cell.…”

Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

“…Preparation of thin sections of trypanosome cells for transmission electron microscopy was carried out according to methods described in our previous publication (32). Briefly, cells were fixed in glutaraldehyde, treated with Millonig’s buffer, and incubated with 2% OsO 4 .…”

Distinct Roles of a Mitogen-Activated Protein Kinase in Cytokinesis between Different Life Cycle Forms of Trypanosoma brucei

“…A 601-bp DNA fragment corresponding to the Cterminal coding region of TbMAPK6 was cloned into the pC-3HA-PAC vector, which was derived from the pC-3HA-BSD vector (30) by replacing the blasticidin S deaminase (BSD) gene with the puromycin N-acetyltransferase (PAC) gene, and the resulting construct was electroporated into the procyclic form 427 cell line and the bloodstream form 221 cell line according to our published procedures (31,32). The pC-TbMAPK6-3HA-PAC construct was also transfected into the pStemLoop-TbMAPK6 bloodstream cell line to monitor the TbMAPK6 protein level after RNAi.…”