Phosphatidylinositol 4-Kinase III-β Is Required for Golgi Maintenance and Cytokinesis in Trypanosoma brucei

Abstract

The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (␣ and ␤). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase ␤ (TbPI4KIII-␤), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-␤ in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-␤ have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-␤ is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

“…Type III PI 4-kinases play crucial roles in cytokinesis in higher eukaryotes (Brill et al, 2000;Rodgers et al, 2007), whereas PtdIns(4,5)P 2 is required for cytokinesis in a variety of cells (Emoto et al, 2005;Field et al, 2005;Janetopoulos and Devreotes, 2006;Wong et al, 2005;Zhang et al, 1999). Thus, the ELC Cdc4p might help link Pik1p and PtdIns(4,5)P 2 synthesis to PtdIns(4,5)P 2 -binding proteins at the contractile ring.…”

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

“…It should be noted that none of the proteins are enriched at the anterior tip of the new FAZ or on the cleavage furrow, making them less likely to be the direct regulators of cytokinesis. These proteins are associated with basal bodies and/or the flagellum (39,41,47) or the microtubule cytoskeleton (53,54) or are localized predominantly in the cytosol (13,43,(48)(49)(50)(51)(52). Like many of the above-mentioned proteins, TbMAPK6 is also distributed throughout the cytosol in both life cycle forms (Fig.…”

“…However, the downstream factors of TbAUK1 and TbPLK in the cytokinesis regulatory pathway(s) have not been identified. In addition to TbAUK1 and TbPLK, a number of other proteins have also been identified as potential regulators of cytokinesis due to the fact that deficiency in these proteins leads to cytokinesis defects (13,39,41,43,(47)(48)(49)(50)(51)(52)(53)(54)(55). Nevertheless, precisely how these proteins are involved in controlling cytokinesis has not been well established.…”

Regulation of the Cell Division Cycle in Trypanosoma brucei

“…Recently, a few proteins have been shown to be essential for cytokinesis in trypanosomes, such as a type III phosphatidylinositol 4-kinase (93), , the protein arginine methyltransferase (27), the small GTPase ARL2 (90), Katanin and Spastin (9), the F-box protein CFB2 (8), a microtubule-associated protein, AIR9 (77), adenylyl cyclases (95), and a kinetoplastid-specific kinesin, TbKIN-C, and its partner kinesin, TbKIN-D (50, 51), but how they are involved in cytokinesis has not been well established. Among these proteins, some are associated with basal bodies and/or the flagellum (72,98), whereas others are localized predominantly in the cytoplasm (27,93) or are involved in regulating microtubule dynamics (9,50,51,90). Since defects in basal body replication/segregation and flagellum attachment unambiguously inhibit cytokinesis in trypanosomes, it is not possible to assign a direct role for these basal body and flagellar proteins in cytokinesis.…”