Inna Goliand

Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.

The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission. Using this approach, ESCRT-III ring and spiral assemblies were resolved and characterized at different stages of abscission. Visualization of IST1 structures in cells lacking the microtubule-severing enzyme spastin and in cells depleted of specific ESCRT-III components or the ATPase VPS4 demonstrated the contribution of these components to the organization and function of ESCRTs in cells. This work provides direct evidence that ESCRT-III proteins form helical filaments to mediate their function in cells and raises new mechanistic scenarios for ESCRT-driven cytokinetic abscission.

Recently the ESCRT-III filamentous complex was designated as the driving force for mammalian cell abscission, that is, fission of the intercellular membrane bridge connecting daughter cells at the end of cytokinesis. However, how ESCRT-III is activated to set on abscission has not been resolved. Here we revisit the role of the upstream canonical ESCRT players ESCRT-II and CHMP6 in abscission. Using high-resolution imaging, we show that these proteins form highly ordered structures at the intercellular bridge during abscission progression. Furthermore, we demonstrate that a truncated version of CHMP6, composed of its first 52 amino acids (CHMP6-N), arrives at the intercellular bridge, blocks abscission, and subsequently leads to cell death. This phenotype is abolished in a mutated version of CHMP6-N designed to prevent CHMP6-N binding to its ESCRT-II partner. Of interest, deleting the first 10 amino acids from CHMP6-N does not interfere with its arrival at the intercellular bridge but almost completely abolishes the abscission failure phenotype. Taken together, these data suggest an active role for ESCRT-II and CHMP6 in ESCRT-mediated abscission. Our work advances the mechanistic understanding of ESCRT-mediated membrane fission in cells and introduces an easily applicable tool for upstream inhibition of the ESCRT pathway in live mammalian cells.

Lysine methylation, catalyzed by protein lysine methyltransferases (PKMTs), is a key player in regulating intracellular signaling pathways. However, the role of PKMTs and the methylation of nonhistone proteins during the cell cycle are largely unexplored. In a recent proteomic screen, we identified that the PKMT SETD6 methylates PLK1-a key regulator of mitosis and highly expressed in tumor cells. In this study, we provide evidence that SETD6 is involved in cell cycle regulation. SETD6-deficient cells were observed to progress faster through the different mitotic steps toward the cytokinesis stage. Mechanistically, we found that during mitosis SETD6 binds and methylates PLK1 on two lysine residues: K209 and K413. Lack of methylation of these two residues results in increased kinase activity of PLK1, leading to accelerated mitosis and faster cellular proliferation, similarly to SETD6-deficient cells. Taken together, our findings reveal a role for SETD6 in regulating mitotic progression, suggesting a pathway through which SETD6 methylation activity contributes to normal mitotic pace.