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.

A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope through microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the “zygotene chromosomal bouquet.” Here, we identified the “zygotene cilium” in oocytes. This cilium provides a cable system for the bouquet machinery and extends throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies and suggests that cilia can control chromosomal dynamics.

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.

Twenty‐five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state‐of‐the‐art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes and is compatible with live‐cell and super‐resolution imaging applications, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein. The protein of interest is thereby site‐specifically fluorescently labeled inside the cell, eliminating the need for protein‐ or peptide‐labeling tags. Whether this labeling approach will change cell biology research is currently unclear, but it clearly has the potential to do so. In this short review, a general overview of this approach is provided, focusing on the imaging of site‐specifically labeled proteins in mammalian tissue culture cells, and highlighting its advantages and limitations for cellular imaging.

We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level.