An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK in<i>Trypanosoma brucei</i>

Lozano-Núñez, Ana; Ikeda, Kyojiro N.; Sauer, Thomas; Graffenried, Christopher L. de

doi:10.1091/mbc.e12-12-0846

Cited by 25 publications

(45 citation statements)

References 58 publications

(102 reference statements)

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“…However, whether TbPLK plays a role in basal body duplication is still controversial. One report suggested that TbPLK is required for basal body duplication (31), whereas other studies showed that TbPLK is required for segregation/rotation, but not duplication, of the basal body (30,32).…”

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confidence: 99%

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

Liu

Zhou

et al. 2015

Eukaryot Cell

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The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.T rypanosoma brucei is an early branching microbial eukaryote and the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. A trypanosome cell possesses a motile flagellum that is nucleated by the basal body, the cell's microtubule organizing center (MTOC), exits the cell body through the flagellar pocket, and is attached to the cell body along most of its length via a specialized cytoskeletal structure termed the flagellum attachment zone (FAZ). The flagellum is composed of a canonical 9 ϩ 2 microtubule axoneme and an extra-axoneme structure termed the paraflagellar rod (PFR), and it is required for cell morphogenesis, cell motility, and cytokinesis (1, 2). Early in the cell cycle, a trypanosome cell possesses a basal body, which nucleates the flagellum axoneme, and an associated probasal body. The probasal body matures to a basal body when the cell proceeds to S phase of the cell cycle; subsequently, two probasal bodies are formed, each of which associates with each of the two mature basal bodies (3, 4). A new flagellum then is assembled from the newly matured basal body, which then undergoes a rotation toward the posterior of the old basal body. Following the elongation of the new flagellum, the new basal body/probasal body pair moves toward the posterior portion of the cell, which constitutes one of the first cytoskeletal events in the cell cycle of T. brucei (3, 4). Movement of the basal body is required for cell morphogenesis (4) and for segregation of the kinetoplast, the cell's mitochondrial genome that is attached to the basal body through a struc...

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confidence: 99%

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

Liu

Zhou

et al. 2015

Eukaryot Cell

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“…TbPLK localization has been shown to be dynamic through the cell cycle. Consistent with its lack of mitotic function, TbPLK does not localize in the nucleus, but is found at the basal bodies, the bilobe structure, the flagellar pocket collar, and the flagellum attachment zone [82][83][84][85][86]. TbPLK regulation, although appearing to be divergent in many aspects, does -in common with other PLKs -require phosphorylation of threonine residues in the activation loop for full kinase activity [87].…”

Section: Polo-like Kinasesmentioning

confidence: 95%

“…Studies characterizing TbPLK function found the kinase to be essential in PCF and BSF T. brucei, but no evidence to date suggests that TbPLK plays a role in mitosis. Instead, TbPLK is essential for basal body rotation and kinetoplast DNA segregation, [80][81][82]83], Golgi biogenesis [84], bilobe duplication via phosphorylation of TbPLK's only known in vivo substrate, Tb Centrin2 [84,85] and flagellum inheritance in PCF parasites [82,83,85] and for furrow ingression during cytokinesis in the BSF stage [81]. TbPLK localization has been shown to be dynamic through the cell cycle.…”

Section: Polo-like Kinasesmentioning

confidence: 99%

Trypanosomatid Cell Division Kinases

Benz

Thomas

Hammarton

2013

Protein Phosphorylation in Parasites

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Advances have been made recently in understanding the order of events in the cell division cycles of Leishmania spp. and Trypanosoma cruzi, adding to previous knowledge of events in Trypanosoma brucei, and highlighting similarities and differences between these trypanosomatids. However, the present understanding of how the cell cycle is regulated by kinases still largely derives from analyses in T. brucei, since convenient genetic tools for analyzing the function of essential regulators -for example, inducible expression systems and/or RNA interferenceare still lacking in Leishmania and T. cruzi. While current understanding of the function of some kinases (e.g., some cyclin-dependent kinases, the NDR kinases, aurora, tousled-like and polo-like kinases) is now very good, many more remain to be characterized, as do details of the signal transduction pathways in which they operate. A number of protein kinases have, in recent years, been genetically -and in some cases also chemically -validated as potential novel drug targets for human African trypanosomiasis or leishmaniasis, although only a few, including CRK3: CYC6, CRK3:CYCA, the NDR kinases, PK50 and PK53 and polo-like kinase, have been subjected to high-throughput screening to identify small-molecule inhibitors. However, problems with these screens include a failure to identify potent inhibitors against the parasite enzyme in vitro, a lack of selectivity for the parasite enzyme over the equivalent human enzyme, or poor efficacy against the parasite. Thus, further chemical investigations will be required to generate new compounds with more ideal properties.

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“…While a few human and T. brucei PKs have residues as small as threonine at this location, the large majority of PKs utilize methionine, leucine or phenylalanine. Although this is not always the case (Oh et al, 2007; Lopez et al, 2014), some PKs tolerate mutation of the gatekeeper residue to small glycine or alanine residues, which enlarges the size of the ATP-binding site (Johnson et al, 2008; Hengeveld et al, 2012; Lourido et al, 2012; Lozano-Nunez et al, 2013). This creates an analog-sensitive PK (as-PK) which can be specifically inhibited by ATP-competitive inhibitors that contain bulky side chains (called bumped kinase inhibitors, BKIs), unlike the wild-type (WT) PK (Liu et al, 1999).…”

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confidence: 99%

“…This chemical genetic approach is similar to therapeutic application of drugs, since the PK is present but inactivated with an ATP-competitive inhibitor, rather than absent as with genetic approaches such as RNA interference (RNAi) or conditional knockouts (cKOs). To our knowledge this approach has been successfully applied in one instance thus far to study the cellular function of a kinetoplastid PK, the polo-like kinase of T. brucei (Lozano-Nunez et al, 2013), but has not yet been used for in vivo studies of parasite infection.…”

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A novel protein kinase is essential in bloodstream Trypanosoma brucei

Jensen

Booster

Vidadala

et al. 2016

International Journal for Parasitology

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Human African trypanosomiasis a fatal disease for which no vaccines exist and treatment regimens are difficult. Here, we evaluate a Trypanosoma brucei protein kinase, AEK1, as a potential drug target. Conditional knockouts confirmed AEK1 essentiality in bloodstream forms. For chemical validation, we overcame the lack of AEK1 inhibitors by creating parasites expressing a single, functional analog-sensitive AEK1 allele. Analog treatment of mice infected with this strain delayed parasitemia and death, with one-third of animals showing no parasitemia. These studies validate AEK1 as a drug target and highlight the need for further understanding of its function.

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An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK inTrypanosoma brucei

Cited by 25 publications

References 58 publications

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

Trypanosomatid Cell Division Kinases

A novel protein kinase is essential in bloodstream Trypanosoma brucei

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