Nuclear Repulsion Enables Division Autonomy in a Single Cytoplasm

SummaryBackgroundCurrent models of cell-cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling. However, synchrony is rarely observed in naturally occurring syncytia, such as the multinucleate fungus Ashbya gossypii. In this system, nuclei divide asynchronously, raising the question of how nuclear timing differences are maintained despite sharing a common milieu.ResultsWe observe that neighboring nuclei are highly variable in division-cycle duration and that neighbors repel one another to space apart and demarcate their own cytoplasmic territories. The size of these territories increases as a nucleus approaches mitosis and can influence cycling rates. This nonrandom nuclear spacing is regulated by microtubules and is required for nuclear asynchrony, as nuclei that transiently come in very close proximity will partially synchronize. Sister nuclei born of the same mitosis are generally not persistent neighbors over their lifetimes yet remarkably retain similar division cycle times. This indicates that nuclei carry a memory of their birth state that influences their division timing and supports that nuclei subdivide a common cytosol into functionally distinct yet mobile compartments.ConclusionsThese findings support that nuclei use cytoplasmic microtubules to establish “cells within cells.” Individual compartments appear to push against one another to compete for cytoplasmic territory and insulate the division cycle. This provides a mechanism by which syncytial nuclei can spatially organize cell-cycle signaling and suggests size control can act in a system without physical boundaries

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth

Morphological traits can track coral reef responses to the Anthropocene

MD was supported by the John Templeton Foundation (60501) and JM was supported by the Australian Research Council (FT110100609) during the period this research was undertaken.1. Susceptibility to human-driven environmental changes is mediated by species traits. Therefore, identifying traits that predict organism performance, ecosystem function and response to changes in environmental conditions can help forecast how ecosystems are responding to the Anthropocene. 2. Morphology dictates how organisms interact with their environment and other organisms, partially determining the environmental and biological contexts in which they are successful. Morphology is important for autogenic ecosystem engineering organisms, such as reef-building corals, because it determines the shape of the structures they create and by extension the communities they support. 3. Here, we present six morphological traits that capture variation in volume compactness, surface complexity and top-heaviness. With support from the literature, we propose causal links between morphology and a performance–function–response framework. 4. To illustrate these concepts, we combine 3D scanning and coral survey data to predict morphological traits from in situ colonies. We present a case study that examines how assemblage-scale morphological traits have responded to two cyclones and the 2016 mass bleaching event—two phenomena predicted to increase in severity in the Anthropocene—and discuss how these changes may impact ecosystem function. 5. The morphological traits outlined here offer a generalised and hypothesis-driven approach to tracking how reefs respond to the Anthropocene. The ability to predict these traits from field data and the increasing use of photogrammetry makes them readily applicable across broad spatiotemporal scales.PostprintPeer reviewe

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.Deutsche Forschungsgemeinschaf

Environmental and biological controls on Mg and Li in deep-sea scleractinian corals

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Earth and Planetary Science Letters 300 (2010): 215-225, doi:10.1016/j.epsl.2010.09.029.Deep-sea scleractinian corals precipitate aragonite skeletons that provide valuable archives of past ocean conditions. During calcification biological mediation causes variability in trace metal incorporation and isotopic ratios of the aragonite such that signals caused by environmental controls can be overwhelmed. This complicates the interpretation of geochemical proxies used for paleo-reconstructions. In this study we examine the environmental controls on the Mg/Li ratio of 34 individuals from seven genera of deep-sea scleractinian corals: Desmophyllum, Balanophyllia, Caryophyllia, Enallopsammia, Flabellum, Trochocyanthus, and Lophelia. In addition we examine the distributions of Mg and Li in Desmophyllum and Balanophyllia using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Both Mg/Ca and Li/Ca ratios increased by more than a factor of 2 in the center of calcification regions compared to the outer, fibrous regions of the coral skeleton. As a result, replicate ~10 mg subsamples of coral show less variability in the Mg/Li ratio than Mg/Ca. Microscale Mg and Li results are consistent with Rayleigh-type incorporation of trace metals with additional processes dominating composition within centers of calcification. Comparison of Mg/Li to seawater properties near the site of collection shows that the ratio is not controlled by either carbonate ion or salinity. It appears that temperature is the major control on the Mg/Li ratio. For all 34 samples the temperature correlation (R2=0.62) is significantly better than for Mg/Ca (R2=0.06). For corals of the family Caryophyllidae the R2 value increases to 0.82 with the exclusion of one sample that was observed to have an altered, chalky texture. Despite this excellent correlation the scatter in the data suggests that the Mg/Li ratio of deep-sea corals cannot be used to reconstruct temperature to better than approximately ±1.6°C without better temperature control and additional calibration points on modern coral samples.Financial Support was provided by the USGS WHOI Co-operative agreement, NSF-ANT grant numbers 0636787 and 80295700 and the WHOI Ocean Life Institute. David Case was supported by the WHOI Summer Student Fellowship

FXR1 splicing is important for muscle development and biomolecular condensates in muscle cells

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Smith, J. A., Curry, E. G., Blue, R. E., Roden, C., Dundon, S. E. R., Rodríguez-Vargas, A., Jordan, D. C., Chen, X., Lyons, S. M., Crutchley, J., Anderson, P., Horb, M. E., Gladfelter, A. S., & Giudice, J. FXR1 splicing is important for muscle development and biomolecular condensates in muscle cells. Journal of Cell Biology, 219(4), (2020): e201911129, doi: 10.1083/jcb.201911129.Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.We thank the A.S. Gladfelter and J. Giudice laboratories, Nancy Kedersha, and Silvia Ramos for critical discussions; Eunice Y. Lee for technical help; Dr. Stephanie Gupton (University of North Carolina at Chapel Hill, Chapel Hill, NC) for donation of WT C57BL/6J mouse embryos; and Marcin Wlizla and National Xenopus Resource (RRID:SCR_013731) for their help in maintaining adult frogs and other important technical support. This work has been funded by a University of North Carolina at Chapel Hill Junior Faculty Development Award (to J. Giudice); a Nutrition and Obesity Research Center, University of North Carolina at Chapel Hill, Pilot & Feasibility Research grant (P30DK056350 to J. Giudice); University of North Carolina at Chapel Hill startup funds (to J. Giudice); the March of Dimes Foundation (5-FY18-36, Basil O’Connor Starter Scholar Award to J. Giudice); and NCTraCs Pilot Grant (550KR181805) from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number UL1TR002489 (to J. Giudice), National Institutes of Health National Institute of General Medical Sciences grants (R01-GM130866 to J. Giudice, R01-GM081506 to A.S. Gladfelter, R35-GM126901 to P. Anderson, K99-GM124458 to S.M. Lyons, R25-GM089569 and 2R25-GM055336-20 to E.G. Curry); Howard Hughes Medical Institute Faculty Scholars program (A.S. Gladfelter), and National Institute of Health grants R01-HD084409 and P40-OD010997 (to M.E. Horb). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.2020-09-1

Regulation of polarised growth in fungi

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasmamembrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24, the GEF for the rho family GTPase, Cdc42. Cdc42 in its GTP-bound activates a multiprotein protein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkörper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkörper. The Spitzenkörper then acts as vesicle supply centre in which vesicles exit the Spitzenkörper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkörper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research

Extreme spatial heterogeneity in carbonate accretion potential on a Caribbean fringing reef linked to local human disturbance gradients

This is the final version. Available on open access from Wiley via the DOI in this recordThe capacity of coral reefs to maintain their structurally complex frameworks and to retain the potential for vertical accretion is vitally important to the persistence of their ecological functioning and the ecosystem services they sustain. However, datasets to support detailed along-coast assessments of framework production rates and accretion potential do not presently exist. Here we estimate, based on gross bioaccretion and bioerosion measures, the carbonate budgets and resultant maximum accretion potential (RAPmax) of the shallow reef zone of leeward Bonaire – between 5 to 12 m depth – at unique fine spatial resolution along this coast (115 sites). Whilst the fringing reef of Bonaire is often reported to be in a better ecological condition than most sites throughout the wider Caribbean region, our data show that the carbonate budgets of the reefs and derived RAPmax rates varied3 considerably across this ~58 km long fringing reef complex. Some areas, in particular the marine reserves, were indeed still dominated by structurally complex coral communities with high net carbonate production (> 10 kg CaCO3 m-2 year-1 35 ), high live coral cover and complex structural topography. The majority of the studied sites, however, were defined by relatively low budget states (< 2 kg CaCO3 m-2 year-1 36 ) or were in a state of net erosion. These data highlight the marked spatial heterogeneity that can occur in budgets states, and thus in reef accretion potential, even between quite closely spaced areas of individual reef complexes. This heterogeneity is linked strongly to the degree of localized land-based impacts along the coast, and resultant differences in the abundance of reef framework building coral species. The major impact of this variability is that those sections of reef defined by low-accretion potential will have limited capacity to maintain their structural integrity and to keep pace with current projections of climate change induced sea-level rise (SLR), thus posing a threat to reef functioning, biodiversity and trophic cascades. Since many Caribbean reefs are more severely degraded than those found around Bonaire, it is to be expected that the findings presented here are rather the rule than the exception, but the study also highlights the need for similar high spatial resolution (along-coast) assessments of budget states and accretion potential to meaningfully explore increasing coastal risk at the country level. The findings also more generally underline the significance of reducing local anthropogenic disturbance and restoring framework-building coral assemblages. Appropriately focussed local preservation efforts may aid in averting future large-scale submergence of Caribbean coral reefs and will constrain the social and economic implications associated with the loss of reef goods and services.Ministry of Economic AffairsWageningen UniversityRoyal Netherlands Institute for Sea Researc

Identification of an Amphipathic Helix Important for the Formation of Ectopic Septin Spirals and Axial Budding in Yeast Axial Landmark Protein Bud3p

Correct positioning of polarity axis in response to internal or external cues is central to cellular morphogenesis and cell fate determination. In the budding yeast Saccharomyces cerevisiae, Bud3p plays a key role in the axial bud-site selection (axial budding) process in which cells assemble the new bud next to the preceding cell division site. Bud3p is thought to act as a component of a spatial landmark. However, it is not clear how Bud3p interacts with other components of the landmark, such as the septins, to control axial budding. Here, we report that overexpression of Bud3p causes the formation of small septin rings (∼1 µm in diameter) and arcs aside from previously reported spiral-like septin structures. Bud3p closely associates with the septins in vivo as Bud3p colocalizes with these aberrant septin structures and forms a complex with two septins, Cdc10p and Cdc11p. The interaction of Bud3p with the septins may involve multiple regions of Bud3p including 1–858, 850–1220, and 1221–1636 a.a. since they all target to the bud neck but exhibit different effects on septin organization when overexpressed. In addition, our study reveals that the axial budding function of Bud3p is mediated by the N-terminal region 1–858. This region shares an amphipathic helix (850–858) crucial for bud neck targeting with the middle portion 850–1103 involved in the formation of ectopic septin spirals and rings. Interestingly, the Dbl-homology domain located in 1–858 is dispensable for axial bud-site selection. Our findings suggest that multiple regions of Bud3p ensure efficient targeting of Bud3p to the bud neck in the assembly of the axial landmark and distinct domains of Bud3p are involved in axial bud-site selection and other cellular processes

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism