Biology subjects

Maddox, A. S.

Publications and source records attributed to Maddox, A. S..

12 recordsLinked to original sources

bioRxiv2026

Tagging C. elegans septins disrupts cytoskeletal scaffolding but not post-embryonic roles

Septins are conserved polymer-forming proteins that scaffold the actomyosin cytoskeleton, its regulators, and other factors to cellular membranes. Septins also sense micron-scale curvature, bind microtubules, and establish membrane diffusion barriers. C. elegans is a powerful animal model to study septins roles because there are only two septin genes: unc-59 and unc-61. In many fungal and animal cell types, septins are required for proper cytokinesis. In the C. elegans zygote, septins scaffolding roles in cytokinesis manifest during the chiral rotation of the cell cortex and the asymmetry of cytokinetic ring closure. Originally named for the uncoordinated movement exhibited by hypomorphic alleles, UNC-59 and UNC-61 are also required for normal postembryonic development, germline development, and fertility. To study C. elegans septins in these various contexts, we sought a fluorescent-protein tagging strategy that minimally perturbed septin function. We examined strains in which GFP, mKate2 or wrmScarlet had been inserted at the unc-59 locus, or coupled to unc-61b/c at an exogenous locus, to encode fluorescently tagged fusion proteins. We compared these tagged septins to classical hypomorphic alleles, and to new null alleles. Null alleles phenocopied hypomorphic alleles in all our assays. Strains bearing fluorescently tagged septins exhibited defects in zygote cytokinesis, qualitatively phenocopying both hypomorphic and null alleles. These findings agreed with recent work with fission yeast, demonstrating the sensitivity of septin function to tagging. Interestingly, tagging septins did not perturb postembryonic development including animal mobility. This suggests that septins play distinct functions in the zygote versus later in development.

cell biology↗

bioRxiv2026

Discovery and in vivo characterization of novel TOG domain-containing proteins using C. elegans

The proper assembly, architecture, and maintenance of microtubule, actin and other cytoskeletal networks require regulation by various polymer binding proteins. Microtubules, rely on both the tubulin building blocks, but also many tubulin- and microtubule binding proteins. TOG domain-containing proteins comprise one family of tubulin-binding proteins that regulate microtubule dynamics. Here we identify two previously uncharacterized TOG domain-containing proteins (TOD-1 and TOD-2) in the nematode, C. elegans. These proteins are unique in that they are members of the XMAP215 family but contain reduced numbers of TOG domains and, in one case, a divergent TOG domain. TOD-1 and TOD-2 are expressed in and contribute to the normal function of sperm. The single TOG domain of TOD-1 and both TOG domains of TOD-2 are predicted to bind free tubulin dimers and not microtubule lattice. Deletion of either tod gene resulted in an increased laying of unfertilized oocytes. Inspection of mutant hermaphrodites revealed a premature onset of sperm migration failure. Together, these findings suggest that C. elegans requires regulation of tubulin dimers and/or microtubules for sperm localization and function. The amoeboid movement of C. elegans sperm has been considered microtubule-independent, our results open a new avenue of research into their unique motility.

cell biology↗

bioRxiv2026

Multiscale temporal tuning of force generation complex machinery governs cortical microtubule interactions during the first mitotic division in C. elegans

Dynein is an essential microtubule motor whose many roles in mitosis complicate efforts to resolve its spatiotemporally dynamic regulation. We previously established a method to classify single particles of the conserved cortical force generation complex (dynein--LIN-5NuMA--GPR-1/2LGN--GGi), as free or interacting with microtubules. Here, we report the results of depleting force generation complex components and regulators. Depleting LIS-1 reversed force asymmetry, while depletion of the regulatory subunit SUR-6PP2A-B55 significantly increased incidence of dynein trajectories with microtubule-interacting behavior during prophase. We next applied our classification scheme to the dynein anchor LIN-5. Microtubule-interacting LIN-5 trajectories were posteriorly enriched during anaphase, consistent with our dynein data and prior fluorescence studies. SUR-6PP2A-B55 depletion did not alter LIN-5 kinetics, suggesting regulation of a dynein specific function rather than regulation via LIN-5. We found evidence that two distinct regulators of force generation complex behavior, microtubule interactions and cortical flows, govern on the millisecond and second time scales, respectively. Our observations provide novel insight into the regulation of cortical dynein and the coupling among the cell membrane, actomyosin cortex, force generation complex, and microtubules that position the mitotic spindle.

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↗

bioRxiv2024

Cellular context specific tuning of actomyosin ring contractility within a common cytoplasm

Actomyosin rings are specializations of the non-muscle actomyosin cytoskeleton that drive cell shape changes during division, wound healing, and other events. Contractile rings throughout phylogeny and in a range of cellular contexts are built from conserved components including non-muscle myosin II, actin filaments, and crosslinking proteins. To explore whether diverse actomyosin rings generate contractile force and close via a common mechanism, we studied three instances of ring closure within the continuous cytoplasm of the C. elegans syncytial oogenic germline: mitotic cytokinesis of germline stem cells, apoptosis of meiotic compartments, and cellularization of oocytes. The three ring types exhibited distinct closure kinetics and component protein abundance dyanmics. We formulated a physical model to relate measured closure speed and molecular composition dynamics to ring active stress and viscosity. We conclude that these ring intrinsic factors vary among the ring types. Our model suggests that motor and non-motor crosslinkers abundance and distribution along filaments are important to recapitulate observed closure dynamics. Thus, our findings suggest that across ring closure contexts, fundamental contractile mechanics are conserved, and the magnitude of contractile force is tuned via regulation of ring component abundance and distribution. These results motivate testable hypotheses about cytoskeletal regulation, architecture, and remodeling.

cell 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↗

bioRxiv2022

Contractile ring composition dictates kinetics of in silico contractility

Constriction kinetics of the cytokinetic ring are expected to depend on dynamic adjustment of ring composition, but the impact of component abundance dynamics on ring constriction is understudied. Computational models generally assume that contractile networks maintain constant composition. To test how compositional dynamics affect constriction kinetics, we first measured F-actin, non-muscle myosin II, septin, and anillin during C. elegans zygotic mitosis. A custom microfluidic device that positioned the cell with the division plane parallel to a light sheet allowed even illumination of the cytokinetic ring. Measured component abundances were implemented in an agent-based 3D model of a membrane-associated contractile ring. With constant network composition, constriction occurred with biologically unrealistic kinetics. Measured changes in component quantities elicited realistic constriction kinetics. Simulated networks were more sensitive to changes in motor and filament amounts, than that of crosslinkers and tethers. Our findings highlight the importance of network composition for actomyosin contraction kinetics. SummaryWe created a microfluidic device compatible with high numerical aperture light sheet microscopy to measure cytokinetic ring component abundance in the C. elegans zygote. Implementing measured dynamics into our three-dimensional agent-based model of a contractile ring elicited biologically realistic kinetics.

cell biology↗

bioRxiv2020

Septins and a formin have distinct functions in chiral cortical rotation in the anaphase C. elegans zygote.

Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the C. elegans zygote actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and crosslink the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation, but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior-posterior axis, and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.

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↗

bioRxiv2017

LITE microscopy: a technology for high numerical aperture, low photobleaching fluorescence imaging

Fluorescence microscopy is a powerful approach for studying sub-cellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel LSFM method: Lateral Interference Tilted Excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically-limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

cell biology↗

bioRxiv2017

Crosslinkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis

Cytokinesis and other cell shape changes are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in non-muscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal crosslinking by myosins and non-motor crosslinkers, are thought to promote contractility. Here, we examined how the abundance of motor and non-motor crosslinkers controls the speed of cytokinetic furrowing. We built a minimal model to simulate the contractile dynamics of the C. elegans zygote cytokinetic ring. This model predicted that intermediate levels of non-motor crosslinkers would allow maximal contraction speed, which we found to be the case for the scaffold protein anillin, in vivo. Our model also demonstrated a non-linear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of non-muscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, both motor and non-motor crosslinkers promote cytokinetic ring closure when present at low levels, but act as a brake when present at higher levels. Together, our findings extend the growing appreciation for the roles of crosslinkers, but reveal that they not only drive but also brake cytoskeletal remodeling.

cell biology↗