The Trypanosoma brucei polo-like kinase homologue is an essential morphogenic regulator of the parasite's cytoskeleton. A series of proteomic screens identifies potential TbPLK binding partners and substrates and better illustrates how the kinase functions, yielding novel proteins involved in flagellar positioning.

The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation identification (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely configured process in kinetoplastids.

The authors note that in the abstract, lines 22-26, "To our knowledge, these results provide the first molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host" were modified to correct an editorial oversight that occurred during the revision of the manuscript. The sentence has been corrected to read "These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host." We apologize for this oversight.Also in the significance statement, lines 1-3, "To our knowledge, this work is the first to identify molecular factors produced by the fungus Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats" has similarly been corrected to read "This work identifies molecular factors produced by the fungus Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats."The online version has been corrected.www.pnas.org/cgi

Trypanosoma brucei uses multiple mechanisms to evade detection by its insect and mammalian hosts. The flagellar pocket (FP) is the exclusive site of uptake from the environment in trypanosomes and shields receptors from exposure to the host. The FP neck is tightly associated with the flagellum via a series of cytoskeletal structures that include the hook complex (HC) and the centrin arm. These structures are implicated in facilitating macromolecule entry into the FP and nucleating the flagellum attachment zone (FAZ), which adheres the flagellum to the cell surface. TbSmee1 (Tb927.10.8820) is a component of the HC and a putative substrate of polo-like kinase (TbPLK), which is essential for centrin arm and FAZ duplication. We show that depletion of TbSmee1 in the insect-resident (procyclic) form of the parasite causes a 40% growth decrease and the appearance of multinucleated cells that result from defective cytokinesis. Cells lacking TbSmee1 contain HCs with aberrant morphology and show delayed uptake of both fluid-phase and membrane markers. TbPLK localization to the tip of the new FAZ is also blocked. These results argue that TbSmee1 is necessary for maintaining HC morphology, which is important for the parasite's ability to take up molecules from its environment.

Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the C. elegans zygote actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and crosslink the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation, but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior-posterior axis, and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan. [Media: see text] [Media: see text]

New methods to directly visualize Rho GTPases reveal how a protein called RhoGDI regulates the activity of these 'molecular switches' at the plasma membrane.

Many cells and tissues exhibit chirality that stems from the chirality of constituent proteins and polymers. For example, the C. elegans zygote undergoes an actomyosin-driven chiral rotation in which the entire cortex is displaced circumferentially around the division plane during anaphase. This phenomenon thus relates to how force and chirality are translated across scales. Although it is known that actomyosin contractility drives this rotation, its molecular mechanisms are incompletely understood. Septins are candidates for contributing to cell-scale chirality due to their ability to anchor and organize the actomyosin cytoskeleton. Here, we report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation, but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to highly abnormal and often reversed cortical rotation. We propose a model by which anaphase cortical contractility is biased in a chiral fashion via interaction between the circumferential cytokinetic ring and perpendicular, longitudinal formin-based actin bundles that have accumulated torsional stress during formin-based polymerization. Our findings thus shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.

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