scholarly journals Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis

2015 ◽  
Vol 26 (3) ◽  
pp. 453-466 ◽  
Author(s):  
Reshma Davidson ◽  
Damien Laporte ◽  
Jian-Qiu Wu
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Nucleotide Exchange ◽  
Protein Levels ◽  
Cellular Processes ◽  
Division Site ◽  
Guanine Nucleotide Exchange ◽  
C Terminus ◽  
Early Anaphase ◽  
Rho Gef

Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. However, the regulation of Rho-GEFs themselves is not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. We show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. As does Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Art1 localization depends on its C-terminus, and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.

scholarly journals The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

2015 ◽  
Vol 26 (2) ◽  
pp. 238-255 ◽  
Author(s):  
Ning Wang ◽  
Mo Wang ◽  
Yi-Hua Zhu ◽  
Timothy W. Grosel ◽  
Daokun Sun ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Cell Polarization ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rho Gef

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.

scholarly journals Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis

2012 ◽  
Vol 23 (7) ◽  
pp. 1181-1195 ◽  
Author(s):  
Yanfang Ye ◽  
I-Ju Lee ◽  
Kurt W. Runge ◽  
Jian-Qiu Wu
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
Division Plane ◽  
Cell Functions ◽  
Division Site ◽  
Ring Assembly ◽  
And Function ◽  
Rho Gef

Cytokinesis is crucial for integrating genome inheritance and cell functions. In multicellular organisms, Rho-guanine nucleotide exchange factors (GEFs) and Rho GTPases are key regulators of division-plane specification and contractile-ring formation during cytokinesis, but how they regulate early steps of cytokinesis in fission yeast remains largely unknown. Here we show that putative Rho-GEF Gef2 and Polo kinase Plo1 coordinate to control the medial cortical localization and function of anillin-related protein Mid1. The division-site positioning defects of gef2∆ plo1-ts18 double mutant can be partially rescued by increasing Mid1 levels. We find that Gef2 physically interacts with the Mid1 N-terminus and modulates Mid1 cortical binding. Gef2 localization to cortical nodes and the contractile ring depends on its last 145 residues, and the DBL-homology domain is important for its function in cytokinesis. Our data suggest the interaction between Rho-GEFs and anillins is an important step in the signaling pathways during cytokinesis. In addition, Gef2 also regulates contractile-ring function late in cytokinesis and may negatively regulate the septation initiation network. Collectively, we propose that Gef2 facilitates and stabilizes Mid1 binding to the medial cortex, where the localized Mid1 specifies the division site and induces contractile-ring assembly.

scholarly journals Cooperation between Rho-GEF Gef2 and its binding partner Nod1 in the regulation of fission yeast cytokinesis

2013 ◽  
Vol 24 (20) ◽  
pp. 3187-3204 ◽  
Author(s):  
Yi-Hua Zhu ◽  
Yanfang Ye ◽  
Zhengrong Wu ◽  
Jian-Qiu Wu
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Adaptor Protein ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
Temporal Regulation ◽  
Division Site ◽  
Septation Initiation Network ◽  
Rho Gef

Cytokinesis is the last step of the cell-division cycle, which requires precise spatial and temporal regulation to ensure genetic stability. Rho guanine nucleotide exchange factors (Rho GEFs) and Rho GTPases are among the key regulators of cytokinesis. We previously found that putative Rho-GEF Gef2 coordinates with Polo kinase Plo1 to control the medial cortical localization of anillin-like protein Mid1 in fission yeast. Here we show that an adaptor protein, Nod1, colocalizes with Gef2 in the contractile ring and its precursor cortical nodes. Like gef2∆, nod1∆ has strong genetic interactions with various cytokinesis mutants involved in division-site positioning, suggesting a role of Nod1 in early cytokinesis. We find that Nod1 and Gef2 interact through the C-termini, which is important for their localization. The contractile-ring localization of Nod1 and Gef2 also depends on the interaction between Nod1 and the F-BAR protein Cdc15, where the Nod1/Gef2 complex plays a role in contractile-ring maintenance and affects the septation initiation network. Moreover, Gef2 binds to purified GTPases Rho1, Rho4, and Rho5 in vitro. Taken together, our data indicate that Nod1 and Gef2 function cooperatively in a protein complex to regulate fission yeast cytokinesis.

scholarly journals The Multiple Functions of Rho GTPases in Fission Yeasts

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1422
Author(s):  
Jero Vicente-Soler ◽  
Teresa Soto ◽  
Alejandro Franco ◽  
José Cansado ◽  
Marisa Madrid
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Model Organism ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Physiological Processes ◽  
Evolutionary Changes ◽  
Rho Family

The Rho family of GTPases represents highly conserved molecular switches involved in a plethora of physiological processes. Fission yeast Schizosaccharomyces pombe has become a fundamental model organism to study the functions of Rho GTPases over the past few decades. In recent years, another fission yeast species, Schizosaccharomyces japonicus, has come into focus offering insight into evolutionary changes within the genus. Both fission yeasts contain only six Rho-type GTPases that are spatiotemporally controlled by multiple guanine–nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and whose intricate regulation in response to external cues is starting to be uncovered. In the present review, we will outline and discuss the current knowledge and recent advances on how the fission yeasts Rho family GTPases regulate essential physiological processes such as morphogenesis and polarity, cellular integrity, cytokinesis and cellular differentiation.

scholarly journals A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion

2001 ◽  
Vol 155 (7) ◽  
pp. 1117-1122 ◽  
Author(s):  
Greg J. Bashaw ◽  
Hailan Hu ◽  
Catherine D. Nobes ◽  
Corey S. Goodman
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Rho Gtpases ◽  
Ectopic Expression ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Guanine Nucleotide Exchange ◽  
Positive Regulators ◽  
The Central Nervous System ◽  
Rho Gef

The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509–514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173–180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103–110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

2012 ◽  
Vol 441 (3) ◽  
pp. 869-880 ◽  
Author(s):  
Erik L. Anderson ◽  
Michael J. Hamann
Keyword(s):  
Rho Gtpases ◽  
Regulatory Protein ◽  
Effector Proteins ◽  
Luciferase Assay ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
P21 Activated Kinase ◽  
Rho Gef ◽  
Split Luciferase

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.

scholarly journals Cdc42 promotes Bgs1 recruitment for septum synthesis and glucanase localization for cell separation during cytokinesis in fission yeast

2019 ◽  
Author(s):  
Udo N. Onwubiko ◽  
Julie Robinson ◽  
Rose Albu Mustaf ◽  
Maitreyi E. Das
Keyword(s):  
Fission Yeast ◽  
Membrane Trafficking ◽  
Cell Separation ◽  
Nucleotide Exchange ◽  
Timely Initiation ◽  
Division Site ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Primary Septum ◽  
Ring Constriction

AbstractCytokinesis in fission yeast involves actomyosin ring constriction concurrent to septum synthesis followed by septum digestion resulting in cell separation. A recent report indicates that endocytosis is required for septum synthesis and cell separation. The conserved GTPase Cdc42 is required for membrane trafficking and promotes endocytosis. Cdc42 is activated by Guanine nucleotide exchange factors (GEFs). Cdc42 GEFs have been shown to promote timely initiation of septum synthesis and proper septum morphology. Here we show that Cdc42 promotes the recruitment of the major primary septum synthesizing enzyme Bgs1 and consequent ring constriction. Cdc42 is also required for proper localization of the septum digesting glucanases at the division site. Thus, Cdc42 is required to promote multiple steps during cytokinesis.

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

2016 ◽  
Vol 27 (8) ◽  
pp. 1235-1245 ◽  
Author(s):  
Bin Wei ◽  
Brian S. Hercyk ◽  
Nicholas Mattson ◽  
Ahmad Mohammadi ◽  
Julie Rich ◽  
...  
Keyword(s):  
Nucleotide Exchange ◽  
Activation Pattern ◽  
Actin Organization ◽  
Cellular Processes ◽  
Septum Formation ◽  
Division Site ◽  
Guanine Nucleotide Exchange ◽  
Ring Assembly ◽  
Ring Constriction ◽  
Type V

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, and scd1Δ cells 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.

scholarly journals Small interfering RNAs as a tool to assign Rho GTPase exchange-factor function in vivo

2002 ◽  
Vol 366 (2) ◽  
pp. 393-398 ◽  
Author(s):  
Alexandra GAMPEL ◽  
Harry MELLOR
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Growth Factor Receptor ◽  
Nucleotide Exchange ◽  
Small Interfering Rnas ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rho Gef

Rho GTPases control a complex network of intracellular signalling pathways. Whereas progress has been made in identifying downstream signalling partners for these proteins, the characterization of Rho upstream regulatory guanine-nucleotide exchange factors (GEFs) has been hampered by a lack of suitable research tools. Here we use small interfering RNAs (siRNAs) to examine the cellular regulation of the RhoB GTPase, and show that RhoB is activated downstream of the epidermal-growth-factor receptor through the Vav2 exchange factor. These studies demonstrate that siRNAs are an ideal research tool for the assignment of Rho GEF function in vivo.

scholarly journals Double inhibition and activation mechanisms of Ephexin family RhoGEFs

2021 ◽  
Vol 118 (8) ◽  
pp. e2024465118
Author(s):  
Meng Zhang ◽  
Lin Lin ◽  
Chao Wang ◽  
Jinwei Zhu
Keyword(s):  
Rho Gtpases ◽  
Fine Tuning ◽  
Inhibition Mechanism ◽  
Binding Motif ◽  
Nucleotide Exchange ◽  
Pdz Proteins ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Activation Mechanisms

Ephexin family guanine nucleotide exchange factors (GEFs) transfer signals from Eph tyrosine kinase receptors to Rho GTPases, which play critical roles in diverse cellular processes, as well as cancers and brain disorders. Here, we elucidate the molecular basis underlying inhibition and activation of Ephexin family RhoGEFs. The crystal structures of partially and fully autoinhibited Ephexin4 reveal that the complete autoinhibition requires both N- and C-terminal inhibitory modes, which can operate independently to impede Ras homolog family member G (RhoG) access. This double inhibition mechanism is commonly employed by other Ephexins and SGEF, another RhoGEF for RhoG. Structural, enzymatic, and cell biological analyses show that phosphorylation of a conserved tyrosine residue in its N-terminal inhibitory domain and association of PDZ proteins with its C-terminal PDZ-binding motif may respectively relieve the two autoinhibitory modes in Ephexin4. Our study provides a mechanistic framework for understanding the fine-tuning regulation of Ephexin4 GEF activity and offers possible clues for its pathological dysfunction.

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