The kinetochore is a macromolecular protein complex that drives chromosome segregation in eukaryotes. Unlike most eukaryotes that have canonical kinetochore proteins, evolutionarily divergent kinetoplastids such as Trypanosoma brucei have unconventional kinetochore proteins. T. brucei also lacks a canonical spindle checkpoint system and it therefore remains unknown how mitotic progression is regulated in this organism. Here we characterized in the procyclic form of T. brucei two paralogous kinetochore proteins with a CLK-like kinase domain, KKT10 and KKT19, which localize at kinetochores in metaphase but disappear at the onset of anaphase. We found that these proteins are functionally redundant. Double knockdown of KKT10/19 led to a significant delay in the metaphase to anaphase transition. We also found that phosphorylation of two kinetochore proteins KKT4 and KKT7 depends on KKT10/19 in vivo. Finally, we showed that the N-terminal part of KKT7 directly interacts with KKT10 and that kinetochore localization of KKT10 depends not only on KKT7 but also on the KKT8 complex. Our results reveal that kinetochore localization of KKT10/19 is tightly controlled to regulate the metaphase to anaphase transition in T. brucei. © 2020. Published by The Company of Biologists Ltd.Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under Gln deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that SNAP29 regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of synaptosome-associated protein 29 (SNAP29) interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation. © 2020. https://www.selleckchem.com/products/sh-4-54.html Published by The Company of Biologists Ltd.The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, a ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted into ILVs. We report that the non-catalytic N terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N terminus inhibits Snf7 association with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and with Vps4, an ATPase recruited by Vps2/Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. These data suggest a mechanism by which the deubiquitination machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling. © 2020. Published by The Company of Biologists Ltd.EML4-ALK is an oncogenic fusion present in ∼5% non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants with different patient outcomes. Here, we show in cell models that EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. It also recruits the NEK9 and NEK7 kinase to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4 as well as constitutive activation of NEK9 also perturb cell morphology and accelerate migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but not ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7 leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer. © 2020. Published by The Company of Biologists Ltd.The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate. © 2020. Published by The Company of Biologists Ltd.