Biology subjects

Werner, M. E.

Publications and source records attributed to Werner, M. E..

5 recordsLinked to original sources

bioRxiv2026

Systematic comparison of genetically encoded F-actin probes for visualizing actomyosin dynamics in vivo using C. elegans

Genetically encoded F-actin probes enable visualization of actin organization and dynamics in living cells, but their performance and effects on cellular function can vary by probe, fluorescent protein, and biological context. Here, we systematically compared three F-actin probes - F-tractin, LifeAct, and Utrophin - fused to mScarlet-I, mNeonGreen, or mStayGold in vivo, using Caenorhabditis elegans. We used single-copy transgenic strains and evaluated fluorescence intensity, photostability, developmental and reproductive outcomes, cytokinetic ring closure, and probe localization across diverse cellular contexts. mStayGold provided the greatest brightness and photostability, making it well suited for long-term time-lapse imaging, whereas mNeonGreen offers an alternative for applications requiring photobleaching. Most of the strains exhibited minimal detectable effects on development and cellular function and outperformed previously generated strains tested. Probe performance varied across tissues and developmental stages. F-tractin generally exhibited lower fluorescence intensity than LifeAct and Utrophin, particularly in embryos. Although the probes largely exhibited subcellular localization patterns consistent with phalloidin staining, differences in their labeling of specific F-actin structures were observed. Together, these results establish a practical framework for selecting genetically encoded F-actin probes for C. elegans, they suggest best candidates for other in vivo systems, and they highlight the importance of considering both fluorescent protein and actin-binding probe when designing live-cell imaging experiments.

cell biology↗

bioRxiv2025

Apoptosis promotes fertility in C. elegans by maintaining functional germline morphology

Programmed cell death (apoptosis) during oogenesis is conserved across metazoans and linked to regulation of oocyte number and quality. In oogenic germlines, the removal of developing oocytes by apoptosis ensures that oocytes do not contain DNA damage or multiple nuclei. Beyond this chromatin-quality control assurance role, it was unknown how apoptosis contributes to oocyte quality. We used the nematode Caenorhabditis elegans (C. elegans) to study the consequences of loss of apoptosis on oogenesis. Blocking apoptosis reduced fecundity in hermaphrodites at peak fertility and caused germline architectural defects such as abnormal rachis morphology and perturbed the arrangement and distribution of oogenic germline compartments. We posit that the defects associated with the loss of germline apoptosis arise due to lack of sufficient space for normal oogonia growth. In support of this idea, oocytes and embryos are abnormally small and exhibit low viability in animals unable to execute apoptosis. These findings suggest that in addition to preventing ploidy defects during oogenesis, apoptosis contributes to fertility by preserving homeostatic germline structure required for the fidelity of oogenesis. Summary StatementBlocking apoptosis during oogenesis disrupts germline structure over time, causing morphological defects and infertility, highlighting the role of apoptosis in maintaining syncytial architecture and reproductive function.

developmental biology↗

bioRxiv2023

Mechanical positive feedback and biochemical negative feedback combine to generate complex contractile oscillations in cytokinesis

Contractile force generation by the cortical actomyosin cytoskeleton is essential for a multitude of biological processes. The actomyosin cortex behaves as an active material that drives local and large-scale shape changes via cytoskeletal remodeling in response to biochemical cues and feedback loops. Cytokinesis is the essential cell division event during which a cortical actomyosin ring generates contractile force to change cell shape and separate two daughter cells. Our recent work with active gel theory predicts that actomyosin systems under the control of a biochemical oscillator and experiencing mechanical strain will exhibit complex spatiotemporal behavior, but cytokinetic contractility was thought to be kinetically simple. To test whether active materials in vivo exhibit spatiotemporally complex kinetics, we used 4-dimensional imaging with unprecedented temporal resolution and discovered sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Quantification of ingression speed oscillations revealed wide ranges of oscillation period and amplitude. In the cytokinetic ring, activity of the master regulator RhoA pulsed with a timescale of approximately 20 seconds, shorter than that reported for any other biological context. Contractility oscillated with 20-second periodicity and with much longer periods. A combination of in vivo and in silico approaches to modify mechanical feedback revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Effective local ring ingression is characterized by slower speed oscillations, likely due to increased local stresses and therefore mechanical feedback. Fast ingression also occurs where material turnover is high, in vivo and in silico. We propose that downstream of initiation by pulsed RhoA activity, mechanical positive feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and therefore makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Our work demonstrates that while biochemical feedback loops afford systems responsiveness and robustness, mechanical feedback must also be considered to describe and understand the behaviors of active materials in vivo.

cell biology↗

bioRxiv2023

Septins throughout phylogeny are predicted to have a transmembrane domain, which in Caenorhabditis elegans is functionally important.

Septins, a conserved family of filament-forming proteins, contribute to eukaryotic cell division, polarity, and membrane trafficking. Septins scaffold other proteins to cellular membranes, but it is not fully understood how septins associate with membranes. We identified and characterized an isoform of Caenorhabditis elegans septin UNC-61 that was predicted to contain a transmembrane domain (TMD). The TMD isoform is expressed in a subset of tissues where the known septins were known to act, and TMD function was required for tissue integrity of the egg-laying apparatus. We found predicted TMD-containing septins across much of opisthokont phylogeny and demonstrated that the TMD-containing sequence of a primate TMD-septin is sufficient for localization to cellular membranes. Together, our findings reveal a novel mechanism of septin-membrane association with profound implications for septin dynamics and regulation.

cell biology↗

bioRxiv2019

Novel cytokinetic ring components limit RhoA activity and contractility

Actomyosin cortical contractility drives many cell shape changes including cytokinetic furrowing. While positive regulation of contractility is well characterized, counterbalancing negative regulation and mechanical brakes are less well understood. The small GTPase RhoA is a central regulator, activating cortical actomyosin contractility during cytokinesis and other events. Here we report how two novel cytokinetic ring components, GCK-1 and CCM-3, participate in a negative feedback loop among RhoA and its cytoskeletal effectors to inhibit contractility. GCK-1 and CCM-3 are recruited by active RhoA and anillin to the cytokinetic ring, where they in turn limit RhoA activity and contractility. This is evidenced by increased RhoA activity, anillin and non-muscle myosin II in the cytokinetic ring, and faster cytokinetic furrowing, following depletion of GCK-1 or CCM-3. GCK-1 or CCM-3 depletion also reduced RGA-3 levels in pulses, and increased baseline RhoA activity and pulsed contractility during zygote polarization. Together, our findings suggest that GCK-1 and CCM-3 regulate cortical actomyosin contractility via negative feedback. SummaryNovel cytokinetic ring proteins, the Ste20 family kinase GCK-1 and its heterodimeric cofactor Cerebral Cavernous Malformations-3, close a negative feedback loop involving the RhoA GAP RGA-3/4, RhoA, and its cytoskeletal effector anillin to limit actomyosin contractility in cytokinesis and during polarization of the C. elegans zygote.

cell biology↗