Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in<i>Trypanosoma brucei</i>

McAllaster, Michael R.; Ikeda, Kyojiro N.; Lozano-Núñez, Ana; Anrather, Dorothea; Unterwurzacher, Verena; Gossenreiter, Thomas; Perry, Jenna A.; Crickley, Robbie; Mercadante, Courtney J.; Vaughan, Sue; Graffenried, Christopher L. de

doi:10.1091/mbc.e15-04-0219

Cited by 77 publications

(145 citation statements)

References 97 publications

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“…S1C), this nomenclature does not sufficiently reflect its localization and function. Although McAllaster et al also showed that RNAi of this protein caused a cytokinesis defect by microscopic inspection, the underlying mechanism was not investigated (23). Our work, however, elucidated the mechanistic role of CIF1 in cytokinesis initiation.…”

Section: Discussionmentioning

confidence: 68%

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Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

Zhou¹,

Zhao

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

160

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Cytokinesis in Trypanosoma brucei, an early branching protozoan, occurs along its longitudinal axis uni-directionally from the anterior tip of the new flagellum attachment zone filament toward the cell's posterior end. However, the underlying mechanisms remain elusive. Here we report that cytokinesis in T. brucei is regulated by a concerted action of Polo-like kinase, Aurora B kinase, and a trypanosome-specific protein CIF1. Phosphorylation of CIF1 by Polo-like kinase targets it to the anterior tip of the new flagellum attachment zone filament, where it subsequently recruits Aurora B kinase to initiate cytokinesis. Consistent with its role, CIF1 depletion inhibits cytokinesis initiation from the anterior end of the cell, but, surprisingly, triggers cytokinesis initiation from the posterior end of the cell, suggesting the activation of an alternative cytokinesis from the opposite cell end. Our results reveal the mechanistic roles of CIF1 and Polo-like kinase in cytokinesis initiation and elucidate the mechanism underlying the recruitment of Aurora B kinase to the cytokinesis initiation site at late anaphase. These findings also delineate a signaling cascade controlling cytokinesis initiation from the anterior end of the cell and uncover a backup cytokinesis that is initiated from the posterior end of the cell when the typical anterior-to-posterior cytokinesis is compromised.cytokinesis | Polo-like kinase | Aurora B kinase | backup cytokinesis |

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Section: Discussionmentioning

confidence: 68%

“…S3). CIF1 was also identified as a substrate of TbPLK in a recent study and was named TOEFAZ1 for Tip Of Extending FAZ 1 (23). However, given its additional localization to the cleavage furrow during cytokinesis (Fig.…”

Section: Discussionmentioning

confidence: 98%

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

Zhou¹,

Zhao

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

160

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“…In contrast, depletion of four FC constituents individually-FCP1, FC1 (also reported in ref. 22), FCP2/TbKinX1, and FCP4/TbKin15-resulted in a substantial increase in cells with their new flagellum tip not in contact with the old flagellum ( Fig. 7 A and B and SI Appendix, Fig.…”

Section: Immunogold Em Labeling Sublocalizes Proteins To Specific Tipmentioning

confidence: 99%

“…Four proteins were present exclusively at the new flagellum tip of dividing 2F cells. These proteins included Tb927.3.4960, a member of the kinetoplastid-specific kinesin-X1 clade (21), which we named FCP2/TbKinX1; Tb927.8.7540, a previously uncharacterized repetitive kinetoplastid-specific protein, FCP3; Tb927.10.890, a member of the ubiquitous kinesin-15 family (21), FCP4/TbKin15; and Tb927.11.1340, which was recently described as the FC constituent FC1 (22). In addition, two proteins without clear homology outside the Kinetoplastida, Tb927.7.6180, which we named axonemal capping structure 1 (ACS1), and Tb927.11.450, ACS2, were present at tips of both new and old flagella (Fig.…”

Section: Structure Immunoprecipitation Approach Identifies Flagellum mentioning

confidence: 99%

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Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

Varga

Moreira-Leite

Portman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The distal end of the eukaryotic flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.

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Identifying Protein‐Protein Interactions by Proximity Biotinylation with AirID and splitAirID

2023

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Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homoor heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes.

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Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis inTrypanosoma brucei

Cited by 77 publications

References 97 publications

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

Identifying Protein‐Protein Interactions by Proximity Biotinylation with AirID and splitAirID

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