spatiotemporal-analysis-of-cytokinetic-events-in-fission-yeast

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

University of Tennessee

It was previously shown that MRG19 downregulates carbon metabolism in Saccharomyces cerevisiae upon glucose exhaustion, and that the gene is glucose repressed. Here, it is shown that glucose repression of MRG19 is overcome upon nitrogen withdrawal, suggesting that MRG19 is a regulator of carbon and nitrogen metabolism. beta-Galactosidase activity fostered by the promoter of GDH1/3, which encode anabolic enzymes of nitrogen metabolism, was altered in an MRG19 disruptant. As compared to the wild-type strain, the MRG19 disruptant showed a decrease in the ratio of 2-oxoglutarate to glutamate under nitrogen-limited conditions. MRG19 disruptants showed reduced pseudohyphal formation and enhanced sporulation, a phenomenon that occurs under conditions of both nitrogen and carbon withdrawal. These studies revealed that MRG19 regulates carbon and nitrogen metabolism, as well as morphogenetic changes, suggesting that MRG19 is a component of the link between the metabolic status of the cell and the corresponding developmental pathway.

Disruption of MRG19 results in altered nitrogen metabolic status and defective pseudohyphal development in Saccharomyces cerevisiae.

Control of cellular dimensions and cell symmetry are critical for development and differentiation. Here we provide evidence that the putative Rho-GAP Rga4p of Schizosaccharomyces pombe controls cellular dimensions. rga4 Delta cells are wider in diameter and shorter in length, whereas Rga4p overexpression leads to reduced diameter of the growing cell tip. Consistent with a negative role in cell growth control, Rga4p protein localizes to the cell sides in a "corset" pattern, and to the nongrowing cell tips. Additionally, rga4 Delta cells show an altered growth pattern similar to that observed in mutants of the formin homology protein For3p. Consistent with these observations, Rga4p is required for normal localization of For3p and for normal distribution of the actin cytoskeleton. We show that different domains of the Rga4p protein mediate diverse morphological functions. The C-terminal GAP domain mediates For3p localization to the cell tips and maintains cell diameter. Conversely, overexpression of the N-terminal LIM homology domain of Rga4p promotes actin cable formation in a For3p-dependent manner. Our studies indicate that Rga4p functionally interacts with For3p and has a novel function in the control of cell diameter and cell growth.

Regulation of cell diameter, For3p localization, and cell symmetry by fission yeast Rho-GAP Rga4p.

To study risk factors for perpetration of spousal violence among men in Bangladesh, self-reported Demographic and Health Surveys data from 2,780 married men using bivariate and multivariate techniques are analyzed. Of the respondents, 74% report having ever been violent toward their wives; 37% report violence in the past year. Nonnormative behaviors increase the risk for violence: Men who marry more than once, men who use drugs, and men who are unfaithful to their wives are all significantly more likely to report violence against their spouses. Egalitarian attitudes toward women do not decrease the risk for violence. However, attitudes explicitly about wife-beating are the strongest predictor of violence: Men who believe wife-beating is acceptable are more than 4 times as likely to report recent violence against their wives. While men's self-reports of spousal violence indicate that wife-beating is prevalent in Bangladesh, the results indicate opportunities for programmatic intervention.

Spousal violence in Bangladesh as reported by men: prevalence and risk factors.

In response to carbon and/or nitrogen limitation, diploid cells of Saccharomyces cerevisiae either sporulate or develop pseudohyphae. Although the signal transduction pathways leading to these developmental changes have been extensively studied, how nutritional signals are integrated is not clearly understood. Results of this study indicate that reducing glucose concentration from 2% (SLAD) to 0.05% (SLALD) causes an increase in the magnitude of filamentation as well as a discernible reduction in the time required for pseudohyphal development. Further, the pseudohyphal defect of gpa2, gpr1and gpa2gpr1 but not the mep2 mutant strain is overcome on SLALD. Low glucose also induced pseudohyphae in mep2gpr1 but not mep2gpa2 strain suggesting that GPR1 inhibits pseudohyphae by inhibiting GPA2 function. Accordingly, deleting GPA2 in mep2gpr1 mutant abrogated pseudohyphae formation in SLALD. Further, replenishment of glucose suppressed pseudohyphal differentiation in wild-type cells grown in SLAD medium. However, in SLALD, glucose replenishment suppressed the filamentation response of gpa2 mutants but not that of strains carrying the wild-type GPA2. Increased trehalose levels correlated with decreased pseudohyphae formation. Results of this study demonstrate that filamentation in response to nitrogen limitation occurs as glucose becomes limiting.

Pseudohyphal differentiation defect due to mutations in GPCR and ammonium signaling is suppressed by low glucose concentration: a possible integrated role for carbon and nitrogen limitation.

The conserved NDR kinase regulates cell morphogenesis and polarized cell growth in different eukaryotic cells ranging from yeast to neurons. Although studies have unraveled the mechanism of regulation of NDR kinase activity, the mechanism of morphology control by NDR and the effectors that mediate NDR function are unknown. Via a chemical genetic approach, we show that the fission yeast NDR homolog, Orb6 kinase, maintains polarized cell growth at the cell tips by spatially regulating the localization of Cdc42 GTPase, a key morphology regulator. Loss of Orb6 kinase activity leads to the recruitment of Cdc42 GTPase and the Cdc42-dependent formin For3, normally found only at the cell tips, to the cell sides. Furthermore, we show that loss of Orb6 kinase activity leads to ectopic lateral localization of the Cdc42 guanine nucleotide exchange factor (GEF) Gef1, but not of the other Cdc42 GEF, Scd1. Consistent with these observations, gef1 deletion suppresses the increased cell diameter phenotype of orb6 mutants. In contrast, the microtubule cytoskeleton and the localization of the microtubule-dependent polarity markers Tea1 and Tea4 are not altered by loss of Orb6 kinase activity. Our findings indicate that the conserved NDR kinase Orb6 regulates cell polarity by spatially restricting the localization and activity of Cdc42 GTPase.

The conserved NDR kinase Orb6 controls polarized cell growth by spatial regulation of the small GTPase Cdc42.

The microtubule cytoskeleton has an important role in the control of mitochondrial distribution in higher eukaryotes. In humans, defects in axonal mitochondrial transport are linked to neurodegenerative diseases. This chapter highlights fission yeast Schizosaccharomyces pombe as a powerful genetic model system for the study of microtubule-dependent mitochondrial movement, dynamics and inheritance.

Microtubule-dependent spatial organization of mitochondria in fission yeast.

Cells promote polarized growth by activation of Rho-family protein Cdc42 at the cell membrane. We combined experiments and modeling to study bipolar growth initiation in fission yeast. Concentrations of a fluorescent marker for active Cdc42, Cdc42 protein, Cdc42-activator Scd1, and scaffold protein Scd2 exhibited anticorrelated fluctuations and oscillations with a 5-minute average period at polarized cell tips. These dynamics indicate competition for active Cdc42 or its regulators and the presence of positive and delayed negative feedbacks. Cdc42 oscillations and spatial distribution were sensitive to the amounts of Cdc42-activator Gef1 and to the activity of Cdc42-dependent kinase Pak1, a negative regulator. Feedbacks regulating Cdc42 oscillations and spatial self-organization appear to provide a flexible mechanism for fission yeast cells to explore polarization states and to control their morphology.

Oscillatory dynamics of Cdc42 GTPase in the control of polarized growth.

Cell polarization is fundamental to many cellular processes, including cell differentiation, cell motility and cell fate determination. A key regulatory enzyme in the control of cell morphogenesis is the conserved Rho GTPase Cdc42, which breaks symmetry via self-amplifying positive-feedback mechanisms. Additional mechanisms of control, including competition between different sites of polarized cell growth and time-delayed negative feedback, define a cellular-level system that promotes Cdc42 oscillatory dynamics and modulates activated Cdc42 intracellular distribution.

Role of Cdc42 dynamics in the control of fission yeast cell polarization.

Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24-Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence.

Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis.

The Rho-family GTPase Cdc42 regulates cell polarity and localizes to the cell division site. Cdc42 is activated by guanine nucleotide exchange factors (GEFs). We report that Cdc42 promotes cytokinesis via a unique spatiotemporal activation pattern due to the distinct action of its GEFs, Gef1 and Scd1, in fission yeast. Before cytokinetic ring constriction, Cdc42 activation, is Gef1 dependent, and after ring constriction, it is Scd1 dependent. Gef1 localizes to the actomyosin ring immediately after ring assembly and promotes timely onset of ring constriction. Gef1 is required for proper actin organization during cytokinesis, distribution of type V myosin Myo52 to the division site, and timely recruitment of septum protein Bgs1. In contrast, Scd1 localizes to the broader region of ingressing membrane during cytokinetic furrowing. Scd1 promotes normal septum formation, andscd1Δcells display aberrant septa with reduced Bgs1 localization. Thus we define unique roles of the GEFs Gef1 and Scd1 in the regulation of distinct events during cytokinesis. Gef1 localizes first to the cytokinetic ring and promotes timely constriction, whereas Scd1 localizes later to the ingressing membrane and promotes septum formation. Our findings are consistent with reports that complexity in GTPase signaling patterns enables exquisite precision over the control of cellular processes.

Unique spatiotemporal activation pattern of Cdc42 by Gef1 and Scd1 promotes different events during cytokinesis.

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

Article	Year
Disruption of MRG19 results in altered nitrogen metabolic status and defective pseudohyphal development in Saccharomyces cerevisiae. Microbiology (Reading, England)\| PubMed ID: 15632429	2005
Regulation of cell diameter, For3p localization, and cell symmetry by fission yeast Rho-GAP Rga4p. Molecular biology of the cell\| PubMed ID: 17377067	2007
Spousal violence in Bangladesh as reported by men: prevalence and risk factors. Journal of interpersonal violence\| PubMed ID: 18523237	2009
Pseudohyphal differentiation defect due to mutations in GPCR and ammonium signaling is suppressed by low glucose concentration: a possible integrated role for carbon and nitrogen limitation. Current genetics\| PubMed ID: 18622617	2008
The conserved NDR kinase Orb6 controls polarized cell growth by spatial regulation of the small GTPase Cdc42. Current biology : CB\| PubMed ID: 19646873	2009
Microtubule-dependent spatial organization of mitochondria in fission yeast. Methods in cell biology\| PubMed ID: 20719273	2010
Oscillatory dynamics of Cdc42 GTPase in the control of polarized growth. Science (New York, N.Y.)\| PubMed ID: 22604726	2012
Role of Cdc42 dynamics in the control of fission yeast cell polarization. Biochemical Society transactions\| PubMed ID: 24256285	2013
Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis. Molecular biology of the cell\| PubMed ID: 26246599	2015
Unique spatiotemporal activation pattern of Cdc42 by Gef1 and Scd1 promotes different events during cytokinesis. Molecular biology of the cell\| PubMed ID: 26941334	2016
Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5. eLife\| PubMed ID: 27474797	2016

Maitreyi Das

Biography

JoVE Journal Publications

Other Publications