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.
Adaptation to the nutritional environment is critical for all cells. RAS GTPase is a highly conserved GTP-binding protein with crucial functions for cell growth and differentiation in response to ...environmental conditions. Here, we describe a novel mechanism connecting RAS GTPase to nutrient availability in fission yeast. We report that the conserved NDR/LATS kinase Orb6 responds to nutritional cues and regulates Ras1 GTPase activity. Orb6 increases the protein levels of an Ras1 GTPase activator, the guanine nucleotide exchange factor Efc25, by phosphorylating Sts5, a protein bound to
mRNA. By manipulating the extent of Orb6-mediated Sts5 assembly into RNP granules, we can modulate Efc25 protein levels, Ras1 GTPase activity, and, as a result, cell growth and cell survival. Thus, we conclude that the Orb6-Sts5-Ras1 regulatory axis plays a crucial role in promoting cell adaptation, balancing the opposing demands of promoting cell growth and extending chronological lifespan.
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.
The conserved NDR kinase regulates cell morphogenesis and polarized cell growth in different eukaryotic cells ranging from yeast to neurons 1–4. Although studies have unraveled the mechanism of ...regulation of NDR kinase activity 4, 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.
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.
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.
The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are ...coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle
Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry‐like Mor2 plays an essential role in ...cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25‐like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1‐Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.
Mitochondrial biogenesis requires the contribution of two genomes and of two compartmentalized protein synthesis systems (nuclear and mitochondrial). Mitochondrial protein synthesis is unique on many ...respects, including the use of a genetic code with deviations from the universal code, the use of a restricted number of transfer RNAs, and because of the large number of nuclear encoded factors involved in assembly of the mitochondrial biosynthetic apparatus. The mitochondrial biosynthetic apparatus is involved in the actual synthesis of a handful of proteins encoded in the mitochondrial DNA. The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are excellent models to identify and study factors required for mitochondrial translation. For that purpose, in vivo mitochondrial protein synthesis, following the incorporation of a radiolabeled precursor into the newly synthesized mitochondrial encoded products, is a relatively simple technique that has been extensively used. Although variations of this technique are well established for studies in S. cerevisiae, they have not been optimized yet for studies in S. pombe. In this chapter, we present an easy, fast and reliable method to in vivo radiolabel mitochondrial translation products from this fission yeast.
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.
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