Asynchronous nuclear division cycles in multinucleated cells

Synchronous mitosis is common in multinucleated cells. We analyzed a unique asynchronous nuclear division cycle in a multinucleated filamentous fungus, Ashbya gossypii. Nuclear pedigree analysis and observation of GFP-labeled spindle pole bodies demonstrated that neighboring nuclei in A. gossypii cells are in different cell cycle stages despite close physical proximity. Neighboring nuclei did not differ significantly in their patterns of cyclin protein localization such that both G1 and mitotic cyclins were present regardless of cell cycle stage, suggesting that the complete destruction of cyclins is not occurring in this system. Indeed, the expression of mitotic cyclin lacking NH(2)-terminal destruction box sequences did not block cell cycle progression. Cells lacking AgSic1p, a predicted cyclin-dependent kinase (CDK) inhibitor, however, showed aberrant multipolar spindles and fragmented nuclei that are indicative of flawed mitoses. We hypothesize that the continuous cytoplasm in these cells promoted the evolution of a nuclear division cycle in which CDK inhibitors primarily control CDK activity rather than oscillating mitotic cyclin proteins

The Effect of Tai Chi Exercise on Balance and Falls in Persons with Parkinson’s

Parkinson’s disease (PD) is a neurodegenerative disease that progresses to impair one’s gait and balance, often causing falls and subsequent disability. Current management of PD is aimed at treating the symptoms but is not effective in treating the underlying cause, nor does typical treatment effectively improve postural stability. Exercise can decrease symptoms of the disease and lessen disability. Providers need to find alternatives to the costly physical therapy that is prescribed to treat progressive and debilitating PD. Tai Chi (TC) has been shown to offer an enjoyable exercise routine that participants want to maintain. There is evidence to support the use of TC as a form of exercise as beneficial in improving balance, reducing falls and promoting quality of life in those with PD (Hackney & Earhart, 2008; Klein & Rivers, 2006; Li et al, 2007). The purpose of this evidence-based practice project was to establish a TC program for persons with PD in a small health system. The Stetler model and the Transtheoretical model of change were used to guide this project. The program developed was 12 weeks in length with two TC sessions of one hour per week. The 20 interested participants that met inclusion criteria were randomized into either the intervention or comparison group. Modified Yang Style TC was taught to 12 persons with PD ages 57 - 89 with Hoehn and Yahr Stages I - IV. All participants also kept an exercise and fall history during the 12 week project. Outcome measures to evaluate balance and quality of life were completed pre and post intervention on both groups and included: a) the Berg Balance Scale, b) the Functional Reach Test, c) the Timed Up and Go, and d) the Parkinson’s Disease Quality of Life Questionairre-39. The data was analyzed using the SPSS 18.0 statistical package. Paired t-tests demonstrated a significant difference in the intervention group for all three pre and post intervention balance measures but did not for falls and quality of life. Findings suggested that TC can be a safe, cost effective exercise for persons with PD to improve balance

Septin Phosphorylation and Coiled-Coil Domains Function in Cell and Septin Ring Morphology in the Filamentous Fungus Ashbya Gossypii

Septins are a class of GTP-binding proteins conserved throughout many eukaryotes. Individual septin subunits associate with one another and assemble into heteromeric complexes that form filaments and higher-order structures in vivo. The mechanisms underlying the assembly and maintenance of higher-order structures in cells remain poorly understood. Septins in several organisms have been shown to be phosphorylated, although precisely how septin phosphorylation may be contributing to the formation of high-order septin structures is unknown. Four of the five septins expressed in the filamentous fungus, Ashbya gossypii, are phosphorylated, and we demonstrate here the diverse roles of these phosphorylation sites in septin ring formation and septin dynamics, as well as cell morphology and viability. Intriguingly, the alteration of specific sites in Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures, implicating septin phosphorylation as a regulator of septin structure formation. Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p causes cell lethality, highlighting the importance of normal septin modification in overall cell function and health. In addition to discovering roles for phosphorylation, we also present diverse functions for conserved septin domains in the formation of septin higher-order structure. We previously showed the requirement for the Shs1p coiled-coil domain in limiting septin ring size and reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and Cdc12p are required for septin ring formation. Our results as a whole reveal novel roles for septin phosphorylation and coiled-coil domains in regulating septin structure and function

Septin Ring Size Scaling and Dynamics Require the Coiled-Coil Region of Shs1p

Septins are conserved GTP-binding proteins that assemble into heteromeric complexes that form filaments and higher-order structures in cells. What directs filament assembly, determines the size of higher-order septin structures, and governs septin dynamics is still not well understood. We previously identified two kinases essential for septin ring assembly in the filamentous fungus Ashbya gossypii and demonstrate here that the septin Shs1p is multiphosphorylated at the C-terminus of the protein near the predicted coiled-coil domain. Expression of the nonphosphorylatable allele shs1-9A does not mimic the loss of the kinase nor does complete truncation of the Shs1p C-terminus. Surprisingly, however, loss of the C-terminus or the predicted coiled-coil domain of Shs1p generates expanded zones of septin assemblies and ectopic septin fibers, as well as aberrant cell morphology. The expanded structures form coincident with ring assembly and are heteromeric. Interestingly, while septin recruitment to convex membranes is increased, septin localization is diminished at concave membranes in these mutants. Additionally, the loss of the coiled-coil leads to increased mobility of Shs1p. These data indicate the coiled-coil of Shs1p is an important negative regulator of septin ring size and mobility, and its absence may make septin assembly sensitive to local membrane curvature

Micron-Scale Plasma Membrane Curvature is Recognized by the Septin Cytoskeleton

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape

Septin assemblies form by diffusion-driven annealing on membranes

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 111 (2014): 2146-2151, doi:10.1073/pnas.1314138111.Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knit together into higher-order assemblies. Using fluorescence correlation spectroscopy (FCS), we determined that cytosolic septins are in small complexes suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence (TIRF) microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends and do not exchange subunits in the middle of filaments. This work shows for the first time that annealing is an intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.This project was supported with funding from by NSF (MCB-507511, ASG) and NIH (GM100160, TT and ASG), and Colwin, Lemann and Spiegel summer fellowships and The Nikon Award for summer investigation at MBL in Woods Hole, MA (ASG) and instrument support from Micro Video Instruments (MVI).2014-07-2

Phosphoregulation provides specificity to biomolecular condensates in the cell cycle and cell polarity

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Gerbich, T. M., McLaughlin, G. A., Cassidy, K., Gerber, S., Adalsteinsson, D., & Gladfelter, A. S. Phosphoregulation provides specificity to biomolecular condensates in the cell cycle and cell polarity. Journal of Cell Biology, 219(7), (2020): e201910021, doi:10.1083/jcb.201910021.Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.The work was supported by National Institutes of Health grant R01-GM-081506

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

Velarium control and visual steering in box jellyfish

Directional swimming in the box jellyfish Tripedalia cystophora (cubozoa, cnidaria) is controlled by the shape of the velarium, which is a thin muscular sheet that forms the opening of the bell. It was unclear how different patterns of visual stimulation control directional swimming and that is the focus of this study. Jellyfish were tethered inside a small experimental tank, where the four vertical walls formed light panels. All four panels were lit at the start of an experiment. The shape of the opening in the velarium was recorded in response to switching off different combinations of panels. We found that under the experimental conditions the opening in the velarium assumed three distinct shapes during a swim contraction. The opening was (1) centred or it was off-centred and pocketed out either towards (2) a rhopalium or (3) a pedalium. The shape of the opening in the velarium followed the direction of the stimulus as long as the stimulus contained directional information. When the stimulus contained no directional information, the percentage of centred pulses increased and the shape of the off-centred pulses had a random orientation. Removing one rhopalium did not change the directional response of the animals, however, the number of centred pulses increased. When three rhopalia were removed, the percentage of centred pulses increased even further and the animals lost their ability to respond to directional information