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Polo-Like Kinase Is Necessary for Flagellum Inheritance In Research Article 3173 Polo-like kinase is necessary for flagellum inheritance in Trypanosoma brucei Kyojiro N. Ikeda1 and Christopher L. de Graffenried2,* 1Department of Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Dr. BohrGasse 9/3, Vienna, Austria 2University of Vienna, Center for Molecular Biology, Max F. Perutz Laboratories, Dr. BohrGasse 9/3, Vienna, Austria *Author for correspondence ([email protected]) Accepted 14 February 2012 Journal of Cell Science 125, 3173–3184 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.101162 Summary Polo-like kinases play an important role in a variety of mitotic events in mammalian cells, ranging from centriole separation and chromosome congression to abscission. To fulfill these roles, Polo-like kinase homologs move to different cellular locations as the cell cycle progresses, starting at the centrosome, progressing to the spindle poles and then the midbody. In the protist parasite Trypanosoma brucei, the single polo-like kinase homolog T. brucei PLK (TbPLK) is essential for cytokinesis and is necessary for the correct duplication of a centrin-containing cytoskeletal structure known as the bilobe. We show that TbPLK has a dynamic localization pattern during the cell cycle. The kinase localizes to the basal body, which nucleates the flagellum, and then successively localizes to a series of cytoskeletal structures that regulate the position and attachment of the flagellum to the cell body. The kinase localizes to each of these structures as they are duplicating. TbPLK associates with a specialized set of microtubules, known as the microtubule quartet, which might transport the kinase during its migration. Depletion of TbPLK causes defects in basal body segregation and blocks the duplication of the regulators that position the flagellum, suggesting that its presence on these structures might be necessary for their proper biogenesis. TbPLK migrates throughout the cell in T. brucei, but the specific locations to which it targets and its functions are geared towards the inheritance of a properly positioned and attached flagellum. Key words: Trypanosoma brucei, PLK, Flagellum, Cell cycle, FAZ, Basal body Introduction TbLRRP1 (Broadhead et al., 2006; Shi et al., 2008; Journal of Cell Science Trypanosoma brucei is the causative agent of sleeping sickness in Selvapandiyan et al., 2007; He et al., 2005; Zhou et al., 2010). A sub-Saharan Africa. It is a flagellated protozoan that requires unique set of four microtubules, known as the microtubule quartet motility in order to evade the immune response of its host (Engstler (MtQ), are nucleated at the basal body, wrap around the flagellar et al., 2007; Hill, 2010). The single flagellum, which is essential pocket, traverse the FPC and then become part of the FAZ, for motility, is nucleated by a basal body near the posterior end of connecting all the structures (Lacomble et al., 2009). the cell and extends towards the anterior (Fig. 1A; the key Taken together these protein complexes regulate the position structures described in this manuscript are also shown). The basal of the flagellum, near its base through the flagellar pocket collar body is adjacent to the kinetoplast, which is the mitochondrial (FPC and bilobe) and along the length of the plasma membrane DNA aggregate that is maintained separate from the nucleus (Gull, (using the bilobe and the FAZ). The proper inheritance of the 1999; Robinson and Gull, 1991). The flagellum is attached to the flagellum requires the coordinated assembly and partitioning of plasma membrane by an underlying cytoskeletal structure known these complexes (Vaughan, 2010; Vaughan and Gull, 2008). This as the flagellum attachment zone (FAZ) (Gull, 1999; Vickerman, has implications for the process of flagellar biogenesis. Flagellum 1969). The basal body is located at the base of an invagination of biogenesis is initiated by the maturation of the probasal body, the cell surface called the flagellar pocket, which is the sole site for which triggers growth of a new flagellum alongside the existing endo- and exo-cytosis (Field and Carrington, 2009; Lacomble et al., one (Fig. 1B, I) (Sherwin and Gull, 1989; Lacomble et al., 2010). 2009). The flagellum emerges from the membrane of the flagellar Once it reaches the flagellar pocket neck, this needs to duplicate, pocket and must therefore exit this compartment. The flagellar so that each flagellar pocket now contains an emergent flagellum. pocket collar (FPC), a structure comprising the protein BILBO1, This requires the duplication of the FPC and the bilobe (Fig. 1B, tightly clinches the neck of the flagellar pocket against the II,III). Further elongation of the new flagellum must then be flagellum, which limits access to the compartment (Bonhivers coupled to growth of a new FAZ, so that the new flagellum is et al., 2008; Gadelha et al., 2009). The neck of the flagellar pocket tethered to the plasma membrane (Fig. 1B, IV). The new is also partially encircled by the hook-like extension of a MORN1- flagellum is also linked to the old one by a structure known as containing cytoskeletal complex, the stem of which coincides with the flagella connector (FC), which guides the positioning of the the posterior part of the FAZ (Morriswood et al., 2009). This new flagellum along the cell body (Moreira-Leite et al., 2001). MORN1 complex is part of a larger cytoskeletal structure termed All this requires the coordinated duplication in sequence of those the bilobe, which also contains small calcium-binding proteins protein complexes that guide the new flagellum to its final called centrins and a leucine-rich repeat protein known as position. What, then, organizes this process? 3174 Journal of Cell Science 125 (13) Fig. 1. Cytoskeletal structures associated with the T. brucei flagellum. (A) A schematic view of the cytoskeletal structures associated with the flagellum. The kinetoplast is present in the posterior of the cell near to the probasal body and basal body, which nucleates the single flagellum. The flagellum emerges from the flagellar pocket, the top of which is tightly clinched by the FPC and bilobe. The flagellum is adhered against the cell body by the FAZ. (B) During the cell cycle, the first structure to duplicate is the basal body. The probasal body matures to nucleate a new flagellum, followed by the formation of two new probasal bodies (I). The pocket and FPC then duplicate, which segregates the new flagellum into its own pocket (II). Subsequently, the bilobe and FAZ duplicate (III). The newly replicated bilobes begin to separate, while the new FAZ continues to extend (IV). As the basal bodies begin to segregate, the kinetoplasts resolve into two structures (V), followed by karyokinesis (VI) and cytokinesis (VII). Journal of Cell Science One of the most important features for any potential regulator of Umeyama and Wang, 2008). In contrast to mammalian PLKs, flagellum positioning is the ability to migrate from one structure to TbPLK does not appear to play a role in mitosis. another to coordinate the assembly of new complexes at precise Here we show that the movement and functioning of TbPLK times as the flagellum is elongating. The polo kinases are can explain the ordered sequence of duplication events of migratory proteins that are known to regulate the biogenesis of the cytoskeletal structures associated with the flagellum. The many cytoskeletal structures in higher eukaryotes. In mammalian migration of the kinase with respect to the basal bodies, bilobe, cells, PLK1 plays important roles in cell division, including FPC and FAZ has been established with high temporal resolution. centriole separation, chromosome congression and cytokinesis TbPLK associates with the new MtQ as it initiates and is present (Barr et al., 2004; Archambault and Glover, 2009). PLKs contain a at the tip of the microtubules as they extend during assembly of phosphopeptide-binding domain that allows them to be targeted to the new FAZ, which could explain how TbPLK migrates through programmed destinations by the action of upstream kinases such as the cell. We show that TbPLK localizes to each structure as it is Cdk1 (Elia et al., 2003a; Elia et al., 2003b; Preisinger et al., 2005). duplicating and that depletion of the kinase prevents duplication, PLK1 is initially found on the centrosome, then migrates to spindle or, in the case of the basal bodies, segregation. Our data show poles and the kinetochores following the breakdown of the nuclear that TbPLK is a vital regulator of key cytoskeletal duplication envelope, and finally localizes to the central spindle and midbody events in T. brucei and that interfering with these highly prior to cytokinesis (Golsteyn et al., 1994; Golsteyn et al., 1995). concerted events leads to irreparable cellular defects. Inhibition of PLK1 function leads to defects in spindle pole formation and chromosome congression (Lane and Nigg, 1996; Results Peters et al., 2006; McInnes et al., 2006). TbPLK migrates from the posterior to the anterior end of In T. brucei, there is only a single PLK homolog, T. brucei the cell and coincides with the duplication of a series of PLK (TbPLK), which we had earlier implicated in the process of cytoskeletal organelles bilobe duplication (de Graffenried et al., 2008). Other evidence Our previous results and those of others showed that during cell suggests that it can localize to the basal bodies and is involved in division TbPLK colocalizes with the basal body, then progresses cytokinesis (Hammarton et al., 2007; de Graffenried et al., 2008; to the bilobe and finally associates with the growing tip of the TbPLK and the flagellum in T. brucei 3175 new FAZ (de Graffenried et al., 2008; Kumar and Wang, 2006; with each marker, along with DIC images, are available in Umeyama and Wang, 2008). TbPLK is present on each of supplementary material Figs S1–3. In an asynchronous culture, these organelles during their duplication phase.
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