Biology subjects

Elting, M. W.

Publications and source records attributed to Elting, M. W..

10 recordsLinked to original sources

bioRxiv2026

Expression of photoactivatable molecules enables FCS in live cells by controlling fluorescence intensity

Cellular systems must act robustly to maintain organismal health, including maintaining biophysical properties that allow for appropriate cellular function, and adapting these properties through changes such as those that occur during cell division. However, we still lack tools to measure many of these physical properties with precision in the living cell. For example, the mechanical properties of the nucleoplasm, the fluid-like substance that fills the nucleus, have not been fully characterized. To investigate these properties, we have turned to the fission yeast Schizosaccharomyces pombe (S. pombe), a well-established, genetically tractable model organism that has been used extensively for studying a variety of cell biophysical processes and structures, including the cytoskeleton and cell division. It is an apt system for studying how the nucleus adapts over the course of the cell cycle, since it undergoes closed mitosis, where the nuclear envelope remains intact during cell division. Studying nucleoplasm properties over the course of closed mitosis may help reveal how nuclear volume, shape, surface area expansion, and chromosome segregation are linked and coordinated. To measure nucleoplasm material properties in S. pombe, we have paired Fluorescence Correlation Spectroscopy (FCS) with a photoswitchable fluorophore, enabling fine control over fluorescent intensity inside live cells. We infer material properties from FCS measurements, while the photoswitchable probe enables confocal imaging in conjunction with these measurements, yielding corresponding information about cellular state and dynamics. Interestingly, we find that nucleoplasm material properties do not vary significantly over the cell cycle. Future studies will use this tool to examine how diverse molecular and genetic perturbations alter nucleoplasmic properties, providing insight into how these properties maintain nuclear function and protect genomic integrity over the cell cycle and during development.

biophysics↗

bioRxiv2026

Spindle biochemistry responds to compressive force from thenuclear envelope to tune spindle dynamics during closed mitosis

During closed mitosis in S. pombe, the nuclear envelope and mitotic spindle must work together mechanically and biochemically to ensure successful nuclear division. Previous work has demonstrated that mechanical force from the nuclear envelope, transmitted through spindle pole bodies, can re-shape the spindle. However, it remains unclear how force might alter spindle biochemistry. Here, we investigate how force reprograms the spindle with two approaches: chronically increasing nuclear envelope tension via the lipid synthesis inhibitor cerulenin, and acutely applying force through an optical trap. Both perturbations slow elongation dynamics and reduce microtubule number. Despite this reduction, key spindle proteins Ase1 and Klp5 increase their density at the spindle midzone, indicating inward force from the nuclear envelope can alter spindle biochemistry. We find that while motor proteins Klp5 and Klp6 only minimally affect the spindles response to increased nuclear envelope force, the combination of removing Ase1 and increasing nuclear envelope force together rescue spindle stability. Together, our findings reveal that nuclear force on the spindle does not merely alter its shape, but is key in regulating its biochemistry to maintain force balance.

biophysics↗

bioRxiv2025

Three-dimensional cellularization in chytrid fungi uses distinct mechanisms from those driving one- and two-dimensional cytokinesis in animals and yeast

Chytrid fungi provide a model for studying foam-like cellularization, where nuclei that are dispersed throughout the cytoplasm are synchronously compartmentalized into daughter cells. This organization poses geometric challenges not faced by cells undergoing conventional cytokinesis or Drosophila monolayer cellularization, where nuclei are organized in linear or planar arrangements with ready-access to the plasma membrane. We use the chytrid Spizellomyces punctatus to show that chytrid cellularization begins with migration of nuclei and their attached centrosomes to the plasma membrane, where centrosome-associated vesicles mark sites of membrane invagination. These vesicles then extend inwards, resulting in tubular furrows that branch and merge to create a honeycomb of polyhedral membrane compartments-a cellularization foam-each with a nucleus and cilium. Using inhibitors and laser ablation, we show that tensile forces produced by actomyosin networks drive aphrogenesis (foam-generation), while microtubules are important for foam patterning and ciliogenesis but are not essential for cellularization. Finally, we suggest that chytrids may have incorporated ancestral mechanisms associated with ciliogenesis to coordinate the association of internal nuclei with membrane furrows to solve the unique geometric challenges associated with aphrogenic cellularization.

cell biology↗

bioRxiv2024

Fishnet mesh of centrin-Sfi1 drives ultrafast calcium-activated contraction of the giant cell Spirostomum ambiguum

Spirostomum is a giant unicellular ciliate that contracts to a quarter of its body length in less than five milliseconds, achieving an order of magnitude higher fractional shortening rate than actomyosin-based systems. This ultrafast contraction is powered by myonemes, calcium-activated protein networks at the cortex whose biochemical mechanism remain unclear. We quantify changes in cortical microtubules, membrane ruffles, and the fishnet-like myoneme mesh during contraction, and develop multiscale models that connect local myoneme shortening to whole-cell shape change. Centrin and an Sfi1 homolog co-localize with the myoneme by immunofluorescence and localize to the myoneme by immunogold electron microscopy. Coarse-grained mesh simulations reproduce the measured deformations and show that fishnet geometry, together with volume conservation, leads to uniform contraction. Finally, we reconstitute a Spirostomum centrin-Sfi1 repeat complex in vitro and measure calcium-dependent compaction and self-association, supporting a molecular basis for myoneme contractility. Together, these results support a multiscale model in which calcium-responsive centrin- Sfi1 structures are the central contractile element in Spirostomum and suggest design principles for fast, calcium-triggered, chemomechanical contractile networks that operate without actomyosin or ATP. SIGNIFICANCE STATEMENTMany cells change shape using actomyosin, but some protists contract using calcium-activated protein networks called myonemes. We combine quantitative imaging, electron microscopy, multiscale modeling, and in vitro reconstitution to link molecular-scale mechanisms to the millisecond shortening of the giant ciliate Spirostomum. Centrin and an Sfi1 homolog co-localize in a fishnet-like cortical mesh, and simulations show that this geometry can reproduce the observed whole-cell shape change under volume conservation. Purified centrin-Sfi1 complexes undergo calcium-dependent compaction and self-association, supporting a protein-scale switch that can drive myoneme contraction. These results connect calcium signaling to whole-cell mechanics and suggest principles for designing fast, ATP-independent bioin-spired actuators and synthetic cellular machinery.

cell biology↗

bioRxiv2022

Pushing the envelope: force balance in fission yeast closed mitosis

The fission yeast S. pombe divides via closed mitosis, meaning that spindle elongation and chromosome segregation transpire entirely within the complete nuclear envelope. Both the spindle and nuclear envelope must undergo significant conformation changes and exert varying forces on each other during this process. Previous work has demonstrated that nuclear envelope expansion1,2 and spindle pole body (SPB) embedding in the nuclear envelope3 are required for normal S. pombe mitosis, and mechanical modeling has described potential contributions of the spindle to nuclear morphology4,5. However, it is not yet fully clear how and to what extent the nuclear envelope and mitotic spindle each directly shape each other during closed mitosis. Here, we investigate this relationship by observing the behaviors of spindles and nuclei in live mitotic fission yeast following laser ablation. First, we characterize these dynamics in molecularly typical S. pombe spindles, finding them to be stabilized by dense crosslinking, before demonstrating that the compressive force acting on the spindle poles is higher in mitotic cells with greater nuclear envelope tension and that spindle compression can be relieved by lessening nuclear envelope tension via laser ablation. Finally, we use a quantitative model to interpret how these data directly demonstrate that fission yeast spindles and nuclear envelopes are a mechanical pair that can each shape the others morphology.

biophysics↗

bioRxiv2022

A unified model for the dynamics of ATP-independent ultrafast contraction

In nature, several ciliated protists possess the remarkable ability to execute ultrafast motions using protein assemblies called myonemes, which contract in response to Ca2+ ions. Existing theories, such as actomyosin contractility and macroscopic biomechanical latches, do not adequately describe these systems, necessitating new models to understand their mechanisms. In this study, we image and quantitatively analyze the contractile kinematics observed in two ciliated protists (Vorticella sp and Spirostomum sp), and, based on the mechanochemistry of these organisms, we propose a minimal mathematical model that reproduces our observations as well as those published previously. Analyzing the model reveals three distinct dynamic regimes, differentiated by the rate of chemical driving and the importance of inertia. We characterize their unique scaling behaviors and kinematic signatures. Besides providing insights into Ca2+-powered myoneme contraction in protists, our work may also inform the rational design of ultrafast bioengineered systems such as active synthetic cells.

biophysics↗

bioRxiv2020

The collapse of the spindle following ablation in S. pombe is mediated by microtubules and the motor protein dynein

A microtubule-based machine called the mitotic spindle segregates chromosomes when eukaryotic cells divide. In the fission yeast S. pombe, which undergoes closed mitosis, the spindle forms a single bundle of microtubules inside the nucleus. During elongation, the spindle extends via antiparallel microtubule sliding by molecular motors. These extensile forces from the spindle are thought to resist compressive forces from the nucleus. We probe the mechanism and maintenance of this force balance via laser ablation of spindles at various stages of mitosis. We find that spindle pole bodies collapse toward each other following ablation, but spindle geometry is often rescued, allowing spindles to resume elongation. While this basic behavior has been previously observed, many questions remain about this phenomenons dynamics, mechanics, and molecular requirements. In this work, we find that previously hypothesized viscoelastic relaxation of the nucleus cannot fully explain spindle shortening in response to laser ablation. Instead, spindle collapse requires microtubule dynamics and is powered at least partly by the minus-end directed motor protein dynein. These results suggest a role for dynein in redundantly supporting force balance and bipolarity in the S. pombe spindle. STATEMENT OF SIGNIFICANCES. pombe serves as an important model organism for understanding cell division. Its structurally simple mitotic spindle is especially suited for mechanical perturbation. Since S. pombe undergoes a process of closed cell division, without breakdown of the nuclear envelope, force may be exerted between its nuclear envelope and spindle. Here, we mechanically sever spindles via laser ablation to probe this force balance. Following ablation, S. pombe spindle fragments collapse toward each other. We find that, contrary to prior expectations, forces from the chromosomes and nuclear envelope are not responsible for this collapse. Instead, it is microtubule-dependent, and is powered at least in part by the minus-end directed microtubule motor protein dynein.

biophysics↗

bioRxiv2020

Automated tracking of S. pombe spindle elongation dynamics

The mitotic spindle is a microtubule-based machine that pulls the two identical sets of chromosomes to opposite ends of the cell during cell division. The fission yeast Schizosaccharomyces pombe is an important model organism for studying mitosis due to its simple, stereotyped spindle structure and well-established genetic toolset. S. pombe spindle length is a useful metric for mitotic progression, but manually tracking spindle ends in each frame to measure spindle length over time is laborious and can limit experimental throughput. We have developed an ImageJ plugin that can automatically track S. pombe spindle length over time and replace manual or semi-automated tracking of spindle elongation dynamics. Using an algorithm that detects the principal axis of the spindle and then finds its ends, we reliably track the length and angle of the spindle as the cell divides. The plugin integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ, and does not require any programming by the user. Thus, the plugin provides an accessible tool for quantification of S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of cell biological perturbations on mitotic progression.

cell biology↗

bioRxiv2020

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes into two eventual daughter nuclei. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) anchor chromosomes within the spindle. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We sever k-fibers using laser ablation, thereby detaching them from poles and testing the contribution of pole-localized force generation to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often, but not always, splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced in response to ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the putative k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15 microtubule binding reduces k-fiber mechanical integrity. In contrast, inhibition of its motor activity but not its microtubule binding does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.

cell biology↗

bioRxiv2017

Mapping load-bearing in the mammalian spindle reveals local kinetochore-fiber anchorage that provides mechanical isolation and redundancy

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore-fibers (k-fibers) to bear this load. We know that the mammalian spindle body can bear the load of chromosome movement far from poles, but do not know where and how - physically and molecularly - this load is distributed across the spindle. In part, this is because perturbing and reading out spindle mechanics in live cells is difficult. Yet, answering this question is key to understanding how the spindle generates and responds to force, and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time, and dissect local anchorage mechanics and mechanism. To do so, we laser ablate single k-fibers at different spindle locations, and in different molecular backgrounds, and quantify at high time resolution the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 m from kinetochores, scaling with k-fiber length, and laterally within ~2 m of k-fiber sides, without neighboring k-fibers sharing load. A phenomenological model constrains the mechanistic underpinnings of these data: it suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement, but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, while Eg5 and PRC1 are not, suggesting specialization in mechanical function and a novel function for NuMA throughout the spindle body. Together, the data and model suggest that widespread NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well-suited to support robust chromosome segregation.

cell biology↗