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.

Mammalian cell abscission was recently shown to be driven by the ESCRT machinery, but the mechanism has not been fully resolved. This work identifies the ESCRT components ESCRT-II and CHMP6 as essential components of ESCRT-mediated abscission and introduces a new approach for inhibition of abscission using the first 52 amino acids of CHMP6.

This work describes an elegant approach for direct, site-specific labeling of proteins with fluorescent-dyes for live cell imaging. By integrating a noncanonical amino acid that is capable of binding a fluorescent dye into tubulin, we directly and specifically labeled tubulin with a fluorescent-dye and imaged microtubules in live mammlian cells.

Studies of ciliopathies have served in elucidating much of our knowledge of structure and function of primary cilia. We report humans with Bardet-Biedl syndrome who display intellectual disability, retinitis pigmentosa, obesity, short stature and brachydactyly, stemming from a homozyogous truncation mutation in SCAPER, a gene previously associated with mitotic progression. Our findings, based on linkage analysis and exome sequencing studies of two remotely related large consanguineous families, are in line with recent reports of SCAPER variants associated with intellectual disability and retinitis pigmentosa. Using immuno-fluorescence and live cell imaging in NIH/3T3 fibroblasts and SH-SY5Y neuroblastoma cell lines over-expressing SCAPER, we demonstrate that both wild type and mutant SCAPER are expressed in primary cilia and co-localize with tubulin, forming bundles of microtubules. While wild type SCAPER was rarely localized along the ciliary axoneme and basal body, the aberrant protein remained sequestered to the cilia, mostly at the ciliary tip. Notably, longer cilia were demonstrated both in human affected fibroblasts compared to controls, as well as in NIH/3T3 cells transfected with mutant versus wildtype SCAPER. As SCAPER expression is known to peak at late G1 and S phase, overlapping the timing of ciliary resorption, our data suggest a possible role of SCAPER in ciliary dynamics and disassembly, also affecting microtubule-related mitotic progression. Thus, we outline a human ciliopathy syndrome and demonstrate that it is caused by a mutation in SCAPER, affecting primary cilia.

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.