A theoretical model of cytokinesis implicates feedback between membrane curvature and cytoskeletal organization in asymmetric cytokinetic furrowing

During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in the Caenorhabditis elegans zygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane–cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity

Cross-linkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis.

Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling

CENP-A and topoisomerase-II antagonistically affect chromosome length

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening

SUMSS: A Wide-Field Radio Imaging Survey of the Southern Sky. I. Science goals, survey design and instrumentation

The Molonglo Observatory Synthesis Telescope, operating at 843 MHz with a 5 square degree field of view, is carrying out a radio imaging survey of the sky south of declination -30 deg. This survey (the Sydney University Molonglo Sky Survey, or SUMSS) produces images with a resolution of 43" x 43" cosec(Dec.) and an rms noise level of about 1 mJy/beam. SUMSS is therefore similar in sensitivity and resolution to the northern NRAO VLA Sky Survey (NVSS; Condon et al. 1998). The survey is progressing at a rate of about 1000 square degrees per year, yielding individual and statistical data for many thousands of weak radio sources. This paper describes the main characteristics of the survey, and presents sample images from the first year of observation.Comment: 27 pages, 12 figures (figures 2, 8, 10 in jpg format); AJ, in pres

Simultaneous Stretching and Contraction of Stress Fibers In Vivo

To study the dynamics of stress fiber components in cultured fibroblasts, we expressed α-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and α-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and α-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider

The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly

In contrast to their sequential roles in midzone assembly, the CPC and centralspindlin act through independent mechanisms to regulate contractile ring assembly

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Model-based trajectory classification of anchored molecular motor-biopolymer interactions

During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA)

Microtubule Dynamics Scale with Cell Size to Set Spindle Length and Assembly Timing.

Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume