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 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

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

Developmental Profiles of Mucosal Immunity in Pre-school Children

This study investigated the effect of attending pre-school on mucosal immunity. Children 3.5 to 5 years of age who attended pre-school were observed for a 10 month period. Demographic information was collected on previous childcare experiences, the home environment and clinical information relating to the child and the family. A daily illness log was kept for each child. A multivariate longitudinal analysis of the relation between immunoglobulins in saliva and age, gender, childcare experience, pre-school exposure, number of siblings, environmental tobacco smoke (ETS), atopy and hospitalisation was conducted. There was a positive association of higher IgA levels with the winter season and with children being older than 4 years (P < .001), having attended childcare prior to commencing pre-school (P < .05), and having been exposed to ETS at home (P < .05). Lower IgA levels were associated with being atopic (P < .05). Higher IgG levels were associated with exposure to ETS (P < .001), while lower levels were associated to having atopy. Higher IgM levels were associated with previous childcare experience (P < .01) whilst having been hospitalised was associated with having low salivary IgM levels (P < .01). Lagged analyses demonstrated that immunological parameters were affected by the number of respiratory infections in the preceding 2 months

Developmental Profiles of Mucosal Immunity in Pre-school Children

This study investigated the effect of attending pre-school on mucosal immunity. Children 3.5 to 5 years of age who attended pre-school were observed for a 10 month period. Demographic information was collected on previous childcare experiences, the home environment and clinical information relating to the child and the family. A daily illness log was kept for each child. A multivariate longitudinal analysis of the relation between immunoglobulins in saliva and age, gender, childcare experience, pre-school exposure, number of siblings, environmental tobacco smoke (ETS), atopy and hospitalisation was conducted. There was a positive association of higher IgA levels with the winter season and with children being older than 4 years (P < .001), having attended childcare prior to commencing pre-school (P < .05), and having been exposed to ETS at home (P < .05). Lower IgA levels were associated with being atopic (P < .05). Higher IgG levels were associated with exposure to ETS (P < .001), while lower levels were associated to having atopy. Higher IgM levels were associated with previous childcare experience (P < .01) whilst having been hospitalised was associated with having low salivary IgM levels (P < .01). Lagged analyses demonstrated that immunological parameters were affected by the number of respiratory infections in the preceding 2 months

Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 27 (2016): 2000-2007, doi:10.1091/mbc.E16-02-0129.Nuclei in syncytia found in fungi, muscles, and tumors can behave independently despite cytoplasmic translation and the homogenizing potential of diffusion. We use a dynactin mutant strain of the multinucleate fungus Ashbya gossypii with highly clustered nuclei to assess the relative contributions of nucleus and cytoplasm to nuclear autonomy. Remarkably, clustered nuclei maintain cell cycle and transcriptional autonomy; therefore some sources of nuclear independence function even with minimal cytosol insulating nuclei. In both nuclear clusters and among evenly spaced nuclei, a nucleus’ transcriptional activity dictates local cytoplasmic contents, as assessed by the localization of several cyclin mRNAs. Thus nuclear activity is a central determinant of the local cytoplasm in syncytia. Of note, we found that the number of nuclei per unit cytoplasm was identical in the mutant to that in wild-type cells, despite clustered nuclei. This work demonstrates that nuclei maintain autonomy at a submicrometer scale and simultaneously maintain a normal nucleocytoplasmic ratio across a syncytium up to the centimeter scale.his work was supported by National Institutes of Health R01-GM081506 (A.S.G., S.E.R., and P.O.), the National Science Foundation GK-12 Program and the Neukom Institute at Dartmouth College (S.E.R.), the Alfred P. Sloan Foundation and National Science Foundation DMS-1351860 (M.R. and S.-S.C.), a National Institutes of Health Ruth L. Kirschstein National Research Service Award (T32-GM008185; S.-S.C.), and the Intramural Research Programs of the National Institutes of Health National Institute of Biomedical Imaging and Bioengineering Whitman Investigator and Grass Foundation Programs at the Marine Biological Laboratory at Woods Hole (A.K. and H.S.

Dissection of Molecular Assembly Dynamics by Tracking Orientation and Position of Single Molecules in Live Cells

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells

Psychological distress and quality of life in lung cancer: The role of health-related stigma, illness appraisals and social constraints

Psycho-Oncology Published by John Wiley &amp; Sons Ltd. Objective Health-related stigma is associated with negative psychological and quality of life outcomes in lung cancer patients. This study describes the impact of stigma on lung cancer patients' psychological distress and quality of life and explores the role of social constraints and illness appraisal as mediators of effect. Methods A self-administered cross-sectional survey examined psychological distress and quality of life in 151 people (59% response rate) diagnosed with lung cancer from Queensland and New South Wales. Health-related stigma, social constraints and illness appraisals were assessed as predictors of adjustment outcomes. Results Forty-nine percent of patients reported elevated anxiety; 41% were depressed; and 51% had high global distress. Health-related stigma was significantly related to global psychological distress and quality of life with greater stigma and shame related to poorer outcomes. These effects were mediated by illness appraisals and social constraints. Conclusions Health-related stigma appears to contribute to poorer adjustment by constraining interpersonal discussions about cancer and heightening feelings of threat. There is a need for the development and evaluation of interventions to ameliorate the negative effects of health-related stigma among lung cancer patients

PolyQ-Dependent RNA–Protein Assemblies Control Symmetry Breaking

Dendritic growth in fungi and neurons requires that multiple axes of polarity are established and maintained within the same cytoplasm. We have discovered that transcripts encoding key polarity factors including a formin, Bni1, and a polarisome scaffold, Spa2, are nonrandomly clustered in the cytosol to initiate and maintain sites of polarized growth in the fungus Ashbya gossypii. This asymmetric distribution requires the mRNAs to interact with a polyQ-containing protein, Whi3, and a Pumilio protein with a low-complexity sequence, Puf2. Cells lacking Whi3 or Puf2 had severe defects in establishing new sites of polarity and failed to localize Bni1 protein. Interaction of mRNAs with Whi3 and Puf2 promotes enrichment of transcripts at established sites of polarized growth and clustering of polarity transcripts throughout the cell body. Thus, aggregation-prone proteins make functional assemblies to position polarity transcripts, and nonrandom positioning of transcripts is required for symmetry-breaking events. This reveals a physiological function for polyQ-driven assemblies in regulating cell polarity