RGS Proteins and Septins Cooperate to Promote Chemotropism by Regulating Polar Cap Mobility

BACKGROUND Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. RESULTS Using microfluidics and live-cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances, the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. CONCLUSIONS Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient.

[1]  Gregory R. Smith,et al.  GTPase-Activating Proteins for Cdc42 , 2002, Eukaryotic Cell.

[2]  R. Goody,et al.  The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation , 2013, eLife.

[3]  J. Pringle,et al.  The septin cortex at the yeast mother-bud neck. , 2001, Current opinion in microbiology.

[4]  Timothy C Elston,et al.  Cellular noise suppression by the regulator of G protein signaling Sst2. , 2014, Molecular cell.

[5]  P. Novick,et al.  Exo84p Is an Exocyst Protein Essential for Secretion* , 1999, The Journal of Biological Chemistry.

[6]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[7]  P B Sigler,et al.  Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast. , 1998, Biochemistry.

[8]  Matthias Peter,et al.  Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating , 2000, Nature Cell Biology.

[9]  E. Bi,et al.  Central Roles of Small GTPases in the Development of Cell Polarity in Yeast and Beyond , 2007, Microbiology and Molecular Biology Reviews.

[10]  B. Haarer,et al.  Role of a Cdc42p effector pathway in recruitment of the yeast septins to the presumptive bud site. , 2005, Molecular biology of the cell.

[11]  Kay Hofmann,et al.  A positive feedback loop stabilizes the guanine‐nucleotide exchange factor Cdc24 at sites of polarization , 2002, The EMBO journal.

[12]  H. Riezman,et al.  A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis. , 2000, Journal of cell science.

[13]  Henrik G. Dohlman,et al.  Pheromone Signaling Mechanisms in Yeast: A Prototypical Sex Machine , 2004, Science.

[14]  P. Kubes,et al.  Neutrophil recruitment and function in health and inflammation , 2013, Nature Reviews Immunology.

[15]  I. Herskowitz,et al.  FAR1 is required for oriented polarization of yeast cells in response to mating pheromones , 1995, The Journal of cell biology.

[16]  Gaudenz Danuser,et al.  Coordination of Rho GTPase activities during cell protrusion , 2009, Nature.

[17]  Y. Barral,et al.  Septins and the lateral compartmentalization of eukaryotic membranes. , 2009, Developmental cell.

[18]  Henrik G. Dohlman,et al.  DEP-Domain-Mediated Regulation of GPCR Signaling Responses , 2006, Cell.

[19]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[20]  E. Elion,et al.  FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1. , 1993, Molecular biology of the cell.

[21]  R. Arkowitz,et al.  Chemical gradients and chemotropism in yeast. , 2009, Cold Spring Harbor perspectives in biology.

[22]  H. Pelham,et al.  Slow Diffusion of Proteins in the Yeast Plasma Membrane Allows Polarity to Be Maintained by Endocytic Cycling , 2003, Current Biology.

[23]  H. Dohlman,et al.  Selective Uncoupling of RGS Action by a Single Point Mutation in the G Protein α-Subunit* , 1998, The Journal of Biological Chemistry.

[24]  M. Snyder,et al.  Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast. , 2000, Molecular cell.

[25]  J. Caviston,et al.  The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast. , 2003, Molecular biology of the cell.

[26]  P. Brennwald,et al.  Spatial regulation of exocytosis and cell polarity: Yeast as a model for animal cells , 2007, FEBS letters.

[27]  J. Derisi,et al.  Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. , 2000, Science.

[28]  L. Giot,et al.  Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromone-induced morphogenesis. , 1997, Molecular biology of the cell.

[29]  Timothy C. Elston,et al.  Tracking Shallow Chemical Gradients by Actin-Driven Wandering of the Polarization Site , 2013, Current Biology.

[30]  J. Thorner,et al.  RGS Proteins and Signaling by Heterotrimeric G Proteins* , 1997, The Journal of Biological Chemistry.

[31]  Chandra L. Theesfeld,et al.  Septin-Dependent Assembly of a Cell Cycle-Regulatory Module in Saccharomyces cerevisiae , 2000, Molecular and Cellular Biology.

[32]  M. Longtine,et al.  Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function , 1998, The Journal of cell biology.

[33]  M. Snyder,et al.  A filamentous growth response mediated by the yeast mating pathway. , 2001, Genetics.

[34]  J. Segall,et al.  Polarization of yeast cells in spatial gradients of alpha mating factor. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Peter,et al.  Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast. , 2000, Molecular cell.

[36]  J. Thorner,et al.  Regulation of G protein-initiated signal transduction in yeast: paradigms and principles. , 2001, Annual review of biochemistry.

[37]  Brian D. Slaughter,et al.  Symmetry breaking in the life cycle of the budding yeast. , 2009, Cold Spring Harbor perspectives in biology.

[38]  Anita T. Layton,et al.  Modeling Vesicle Traffic Reveals Unexpected Consequences for Cdc42p-Mediated Polarity Establishment , 2011, Current Biology.

[39]  Andrew B. Goryachev,et al.  Daughter Cell Identity Emerges from the Interplay of Cdc42, Septins, and Exocytosis , 2013, Developmental cell.

[40]  Stephan Saalfeld,et al.  Software for bead-based registration of selective plane illumination microscopy data , 2010, Nature Methods.

[41]  Tau-Mu Yi,et al.  Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction , 2013, Molecular biology of the cell.

[42]  I. Herskowitz,et al.  The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating. , 1998, Science.

[43]  E. Bi,et al.  Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein–mediated zone of inhibition , 2007, The Journal of cell biology.

[44]  Eugenio Marco,et al.  Endocytosis Optimizes the Dynamic Localization of Membrane Proteins that Regulate Cortical Polarity , 2007, Cell.

[45]  J. Hasty,et al.  Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5. , 2008, Molecular cell.

[46]  M. Snyder,et al.  Bud-site selection and cell polarity in budding yeast. , 2002, Current opinion in microbiology.

[47]  E. Bi,et al.  Cell Polarization and Cytokinesis in Budding Yeast , 2012, Genetics.

[48]  Aljoscha Nern,et al.  A Cdc24p-Far1p-Gβγ Protein Complex Required for Yeast Orientation during Mating , 1999, The Journal of cell biology.

[49]  Amy S. Gladfelter,et al.  Interplay between septin organization, cell cycle and cell shape in yeast , 2005, Journal of Cell Science.