The RACK1 Homologue from Trypanosoma brucei Is Required for the Onset and Progression of Cytokinesis

Abstract

The receptor for activated C kinase 1 (RACK1) is a conserved scaffold protein that helps regulate a range of cell activities including cell growth, shape, and protein translation. We report that a homologue of RACK1 is required for cytokinesis in pathogenic Trypanosoma brucei. The protein, referred to as TRACK, is comprised of WD repeat elements and can complement cpc2 null mutants of Schizosaccharomyces pombe. TRACK is expressed throughout the trypanosome life cycle and is distributed predominantly in a perinuclear region and the cytoplasm but not along the endoplasmic reticulum, mitochondrion, or cleavage furrow of dividing cells. When tetracycline-inducible RNA interference (RNAi) is used to deplete the cellular content of TRACK, the cells remain metabolically active, but growth is inhibited. In bloodstream forms, growth arrest is due to a delay in the onset of cytokinesis. By contrast, procyclic forms are able to initiate cytokinesis in the absence of TRACK but arrest midway through cell cleavage. The RNAi cells undergo multiple rounds of partial cytokinesis and accumulate nuclei and cytoplasmic extensions with attached flagella. The TRACK RNAi construct is also inducible within infected mice. Under these conditions parasites are eliminated from peripheral blood within 3 days post-infection. Taken as a whole, these data indicate that trypanosomes utilize a RACK1 homologue to regulate the final stages of mitosis. Moreover, disrupting the interaction between TRACK and its partners might be targeted in the design of novel therapies.African trypanosomes are protozoan parasites that produce lethal infections in humans and livestock throughout sub-Saharan Africa. Trypanosomes have a complex life cycle that involves changes in morphology, biochemistry, and gene expression as the cells pass through a variety of different environments. Signal cascades are predicted to mediate complex life cycle events and initiate rapid changes in cell behavior. It is hypothesized that ablation of a signal or induction of an inappropriate signal will be lethal to these cells. From past experiments it is clear that trypanosomes have the potential to propagate signals involving Ca 2ϩ , cyclic nucleotides, eicosenoic acid, and phosphoryl transfer (1-4). However, in stark contrast with mammalian cells, where complex interacting signal networks have been described, a simple signal pathway has yet to be identified in Trypanosoma brucei. To remedy this situation we began to study a putative signal anchor protein and the pathways it regulates.In general, anchor proteins help provide spatial organization to the signal process by allowing the formation of multimeric complexes at the appropriate location in the cell (5). Additionally, association with different anchors allows some signal kinases to participate in and distinguish between multiple pathways. The receptor for activated C kinase-1 (RACK1 2 is a WD repeat protein that forms ternary complexes with a range of signal proteins (for review, see Ref. 6). Target proteins interact with RACK1...

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

“…Presumably, these cells did not initiate cytokinesis, which heavily depends on correct mitochondrial DNA segregation and flagellar attachment (LaCount et al, 2002;Robinson and Gull, 1991), but re-replicated their DNA, as has previously been reported (Hammarton et al, 2007;LaCount et al, 2002). This uncoupling of mitosis and cytokinesis is consistent with reports showing that the cytokinesis checkpoint present in other eukaryotes is not functional in trypanosomes (Ellis et al, 2004;Grellier et al, 1999;Ngo et al, 1998;Rothberg et al, 2006). The number of parasites with multiple DNA and flagellar content increased after prolonged TbSPT2 RNAi induction or treatment with high concentrations of myriocin.…”

“…A pleomorphic phenotype results from perturbations of several cell cycle events. At early timepoints, we observed dividing cells with a stable cleavage furrow that persisted through DNA replication, a phenotype similar to that reported in T. brucei for receptor for activated C kinase 1 (TRACK1) RNAi lines (Rothberg et al, 2006). In others, cytokinesis was completed, but mispositioning of the cleavage furrow resulted in anucleate cells and their complements.…”

“…Asynchronous DNA replication and segregation could be observed across the cleavage furrow (Fig. 4Cii), very similar to a phenotype previously reported in T. brucei for RNAi of the receptor for activated C kinase 1 (Rothberg et al, 2006). Because of aberrant cytokinesis, asynchronous DNA replication, and delay in kinetoplast division, XNXK cells did not always contain equal numbers of nuclei and kinetoplasts (Fig.…”

Fission Yeast Receptor of Activated C Kinase (RACK1) Ortholog Cpc2 Regulates Mitotic Commitment through Wee1 Kinase

“…For example, in the parasite Trypanosoma brucei, TRACK (RACK1 ortholog) participates in the control of the final stages of mitosis and cytokinesis (28). Also, genetic and biochemical approaches have shown that RACK1 negatively regulates cell cycle progression at the G 1 /S transition in mammalian cells by suppressing the activity of Src, a nonreceptor protein tyrosine kinase that plays a multitude of roles in cell signaling, and Src-mediated cell cycle regulators in G 1 , thereby promoting a delay of entry into S phase (29,30).…”

RACK1 expression contributes to JNK activity, but JNK activity does not enhance RACK1 expression in hepatocellular carcinoma SMMC-7721 cells

“…Receptor for activated C kinase 1 (RACK1), coded for by the GNB2L1 gene, is a scaffold protein with a propeller-like structure of seven WD40 repeats (6)(7)(8)(9)(10). Numerous studies have suggested that RACK1 plays a pivotal role in the coordination of cell growth, migration and differentiation during tumorigenesis (6)(7)(8)(9)(10).…”