Trypanosomes have divergent kinesin-2 proteins that function differentially in IFT, cell division, and motility

Abstract

24Trypanosoma brucei, the causative agent of African sleeping sickness, has a flagellum 25 that is crucial for motility, pathogenicity, and viability. In most eukaryotes, the intraflagellar 26 transport (IFT) machinery drives flagellum biogenesis, and anterograde IFT requires kinesin-2 27 motor proteins. In this study, we investigated the function of the two T. brucei kinesin-2 proteins, 28TbKin2a and TbKin2b, in bloodstream form trypanosomes. We found that compared to other 29 kinesin-2 proteins, TbKin2a and TbKin2b show greater variation in neck, stalk, and tail domain 30 sequences. Both kinesins contributed additively to flagellar lengthening. Surprisingly, silencing 31TbKin2a inhibited cell proliferation, cytokinesis and motility, whereas silencing TbKin2b did 32 not. TbKin2a was localized on the flagellum and colocalized with IFT components near the basal 33 body, consistent with it performing a role in IFT. TbKin2a was also detected on the flagellar 34 attachment zone, a specialized structure in trypanosome cells that connects the flagellum to the 35 cell body. Our results indicate that kinesin-2 proteins in trypanosomes play conserved roles in 36 IFT and exhibit a specialized localization, emphasizing the evolutionary flexibility of motor 37 protein function in an organism with a large complement of kinesins. 38 -3 -

A Grow-and-Lock Model for the Control of Flagellum Length in Trypanosomes

“…To reduce IFT trafficking, we selected to deplete the expression of kinesin II, the IFT anterograde motor. The genome of T. brucei encodes two putative kinesin II proteins (KIN2A TriTrypDB Tb927.5.2090 and KIN2B TriTrypDB Tb927.11.13920) but no kinesin-associated protein (KAP) [30,31]. Individual RNAi silencing of KIN2A or KIN2B did not result in a visible phenotype: cells assembled apparently normal flagella and grew normally in culture (data not shown), suggesting redundancy.…”

“…Hence, simultaneous knockdown of KIN2A and KIN2B was performed following stable transformation of trypanosomes with a plasmid expressing doublestranded RNA (dsRNA) of both KIN2A and KIN2B under the control of tetracycline-inducible promoters [32]. The efficiency of RNAi silencing in KIN2A2B RNAi cells was confirmed by western blotting using an antibody against KIN2B [31] ( Figure S1). The signal for KIN2B dropped by at least 8-fold from day 1 and remained low for at least 6 days, confirming the efficiency of RNAi silencing ( Figure S1).…”

“…Proteins were transferred overnight at 25V at 4 C to polyvinylidene fluoride membranes (PVDF), then blocked with 5% skimmed milk in PBS-Tween 0.1% (PBST) and incubated with primary antibodies diluted in 1% milk and PBST. The anti-KIN2B (a kind gift of Robert L. Douglas, Berkeley) [31] serum was diluted 1/100. As loading controls, antibodies against ALBA proteins [60] diluted 1/500 were used.…”