scholarly journals ALS2 regulates endosomal trafficking, postsynaptic development, and neuronal survival

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Joohyung Kim ◽  
Sungdae Kim ◽  
Minyeop Nahm ◽  
Tsai-Ning Li ◽  
Hsin-Chieh Lin ◽  
...  
Keyword(s):  
Neuronal Survival ◽  
Small Gtpase ◽  
Structural Defects ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Motor Neuron Diseases ◽  
Endosome Trafficking ◽  
C Terminus ◽  
Nucleotide Exchange Factor ◽  
Locomotor Impairment

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. Here, we demonstrate that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). We further demonstrate that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival.

scholarly journals Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2089 ◽  
Author(s):  
Iker Lamas ◽  
Nathalie Weber ◽  
Sophie G. Martin
Keyword(s):  
Fission Yeast ◽  
Positive Feedback ◽  
Antagonistic Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Architecture

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

scholarly journals Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5

2008 ◽  
Vol 19 (7) ◽  
pp. 2718-2728 ◽  
Author(s):  
Irfan J. Lodhi ◽  
Dave Bridges ◽  
Shian-Huey Chiang ◽  
Yanling Zhang ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Critical Role ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain–containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.

scholarly journals Myoblast Migration and Directional Persistence Affected by Syndecan-4-Mediated Tiam-1 Expression and Distribution

2020 ◽  
Vol 21 (3) ◽  
pp. 823 ◽  
Author(s):  
Daniel Becsky ◽  
Szuzina Gyulai-Nagy ◽  
Arpad Balind ◽  
Peter Horvath ◽  
Laszlo Dux ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Underlying Mechanism ◽  
Small Gtpase ◽  
Asymmetric Distribution ◽  
Nucleotide Exchange ◽  
Muscle Stem Cells ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
T Lymphoma ◽  
Migration Capacity

Skeletal muscle is constantly renewed in response to injury, exercise, or muscle diseases. Muscle stem cells, also known as satellite cells, are stimulated by local damage to proliferate extensively and form myoblasts that then migrate, differentiate, and fuse to form muscle fibers. The transmembrane heparan sulfate proteoglycan syndecan-4 plays multiple roles in signal transduction processes, such as regulating the activity of the small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) by binding and inhibiting the activity of Tiam1 (T-lymphoma invasion and metastasis-1), a guanine nucleotide exchange factor for Rac1. The Rac1-mediated actin remodeling is required for cell migration. Syndecan-4 knockout mice cannot regenerate injured muscle; however, the detailed underlying mechanism is unknown. Here, we demonstrate that shRNA-mediated knockdown of syndecan-4 decreases the random migration of mouse myoblasts during live-cell microscopy. Treatment with the Rac1 inhibitor NSC23766 did not restore the migration capacity of syndecan-4 silenced cells; in fact, it was further reduced. Syndecan-4 knockdown decreased the directional persistence of migration, abrogated the polarized, asymmetric distribution of Tiam1, and reduced the total Tiam1 level of the cells. Syndecan-4 affects myoblast migration via its role in expression and localization of Tiam1; this finding may facilitate greater understanding of the essential role of syndecan-4 in the development and regeneration of skeletal muscle.

Integrin-Independent Role of CalDAG-GEFI in Neutrophil Chemotaxis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1266-1266 ◽  
Author(s):  
Carla Carbo ◽  
Tobias Goerge ◽  
Hidenori Hattori ◽  
Daniel Duerschmied ◽  
Stephen M. Cifuni ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Small Gtpase ◽  
Calcium Flux ◽  
Migration Speed ◽  
Neutrophil Chemotaxis ◽  
Nucleotide Exchange ◽  
Transwell Assay ◽  
Nucleotide Exchange Factor

Abstract Neutrophil chemotaxis and transmigration towards a source of inflammation are two crucial processes for host defense against infection that rely on integrin function. Recently, integrin-independent migration of dendritic cells to the lymph node has been brought to light, although neutrophil migration in the presence of EDTA was reported many years ago. Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is a small signaling protein that plays a key role in the activation of beta-1, beta-2, and beta-3 integrins in platelets and neutrophils by activating the small GTPase Rap1. We explored the role of CalDAG-GEFI in integrin-independent chemotaxis in neutrophils. Here we report that CalDAG-GEFI−/− neutrophils have impaired chemotaxis that is independent of integrin function. In a chemotaxis transwell assay towards LTB4 and in the presence of 10mM EDTA, CalDAG-GEFI−/− neutrophils had a 50% reduction in transmigration over 60 minutes compared to wild-type (WT) neutrophils (p<0.05). In separate experiments we confirmed that the transwell assay is independent of integrins using either CD18−/− neutrophils or WT neutrophils plus a blocking anti-CD18 monoclonal antibody. We previously showed that LTB4 signaling upstream of CalDAG-GEFI was not affected in CalDAG-GEFI−/− neutrophils, as assessed by intracellular calcium flux measurements. Using videomicroscopy to visualize the live migrating neutrophils in a horizontal plate in the presence of 10mM EDTA, we found that the reason CalDAG-GEFI−/− neutrophils fail to reach the chemotactic stimulus (10 pg/mL LTB4) is because they have a significantly reduced migration speed compared to WT neutrophils (16 um/sec vs. 23 um/sec, p<0.05), and also because they have an abnormal chemotactic directionality, with a directionality index (the distance between the start and finish points of a migrating neutrophil/total distance covered by the migrating neutrophil) of 0.84 vs 0.94 in WT neutrophils, p<0.05. We investigated whether the observed differences in chemotaxis between CalDAG-GEFI−/− and WT neutrophils could be explained by differences in F-actin polymerization. Using fluorescence microscopy, we found that the percentage of CalDAG-GEFI−/− neutrophils with F-actin pseudopodia after LTB4 stimulation was significantly lower compared to WT neutrophils (22% vs. 56.7%, p<0.05), suggesting that CalDAG-GEFI−/− neutrophils have a defect in F-actin polymerization. Overall, our studies suggest that CalDAG-GEFI plays a role in the mechanisms that regulate both the migration speed and direction of neutrophils during chemotaxis, independent of its established role in integrin activation.

scholarly journals Semaphorin 4D activates the MAPK pathway downstream of plexin-B1

2006 ◽  
Vol 394 (2) ◽  
pp. 459-464 ◽  
Author(s):  
Jennifer Aurandt ◽  
Weiquan Li ◽  
Kun-Liang Guan
Keyword(s):  
Nervous System ◽  
Mapk Pathway ◽  
Large Family ◽  
Cellular Systems ◽  
Mitogen Activated Protein Kinase ◽  
Nucleotide Exchange ◽  
Semaphorin 4D ◽  
C Terminus ◽  
Nucleotide Exchange Factor ◽  
Mitogen Activated Protein

Semaphorins are a large family of transmembrane and secreted proteins that signal primarily through the receptor plexin. Semaphorins have been characterized in the nervous system as axon guidance cues; however, they have also been shown to control development of other cellular systems such as the vasculature and lungs. As the role of semaphorins outside of the nervous system has broadened, so has elucidation of the intracellular signalling pathways they initiate. Previously, we and others have shown that plexin-B1 activates RhoA through the binding and activation of RhoGEF (guanine nucleotide-exchange factor)/LARG (leukaemia-associated RhoGEF) in response to semaphorin 4D stimulation. In the present study, we show that semaphorin 4D activates the MAPK (mitogen-activated protein kinase) pathway. We have found that the mechanism of activation requires the C-terminus of plexin-B1 and the activation of RhoA.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

scholarly journals The Nuclear RhoA Exchange Factor Net1 Interacts with Proteins of the Dlg Family, Affects Their Localization, and Influences Their Tumor Suppressor Activity

2007 ◽  
Vol 27 (24) ◽  
pp. 8683-8697 ◽  
Author(s):  
Rafael García-Mata ◽  
Adi D. Dubash ◽  
Lisa Sharek ◽  
Heather S. Carr ◽  
Jeffrey A. Frost ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Binding Motif ◽  
Nucleotide Exchange ◽  
Suppressor Activity ◽  
Pdz Proteins ◽  
Exchange Factor ◽  
Tumor Suppressor Proteins ◽  
C Terminus ◽  
Nucleotide Exchange Factor ◽  
Tumor Suppressor Activity

ABSTRACT Net1 is a RhoA-specific guanine nucleotide exchange factor which localizes to the nucleus at steady state. A deletion in its N terminus redistributes the protein to the cytosol, where it activates RhoA and can promote transformation. Net1 contains a PDZ-binding motif at the C terminus which is essential for its transformation properties. Here, we found that Net1 interacts through its PDZ-binding motif with tumor suppressor proteins of the Dlg family, including Dlg1/SAP97, SAP102, and PSD95. The interaction between Net1 and its PDZ partners promotes the translocation of the PDZ proteins to nuclear subdomains associated with PML bodies. Interestingly, the oncogenic mutant of Net1 is unable to shuttle the PDZ proteins to the nucleus, although these proteins still associate as clusters in the cytosol. Our results suggest that the ability of oncogenic Net1 to transform cells may be in part related to its ability to sequester tumor suppressor proteins like Dlg1 in the cytosol, thereby interfering with their normal cellular function. In agreement with this, the transformation potential of oncogenic Net1 is reduced when it is coexpressed with Dlg1 or SAP102. Together, our results suggest that the interaction between Net1 and Dlg1 may contribute to the mechanism of Net1-mediated transformation.

scholarly journals Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

2009 ◽  
Vol 29 (12) ◽  
pp. 3297-3306 ◽  
Author(s):  
Adam Mor ◽  
Joseph P. Wynne ◽  
Ian M. Ahearn ◽  
Michael L. Dustin ◽  
Guangwei Du ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Bound State ◽  
Small Gtpase ◽  
Resting Cells ◽  
Nucleotide Exchange ◽  
Lymphocyte Adhesion ◽  
Nucleotide Exchange Factor ◽  
Phospholipase D1

ABSTRACT Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.

scholarly journals A GAP that Divides

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1788 ◽  
Author(s):  
Angika Basant ◽  
Michael Glotzer
Keyword(s):  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
C Elegans ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Parallel Pathway ◽  
Mutant Phenotypes

Cytokinesis in metazoan cells is mediated by an actomyosin-based contractile ring that assembles in response to activation of the small GTPase RhoA. The guanine nucleotide exchange factor that activates RhoA during cytokinesis, ECT-2, is highly regulated. In most metazoan cells, with the notable exception of the early Caenorhabditis elegans embryo, RhoA activation and furrow ingression require the centralspindlin complex. This exception is due to the existence of a parallel pathway for RhoA activation in C. elegans. Centralspindlin contains CYK-4 which contains a predicted Rho family GTPase-activating protein (GAP) domain. The function of this domain has been the subject of considerable debate. Some publications suggest that the GAP domain promotes RhoA activation (for example, Zhang and Glotzer, 2015; Loria, Longhini and Glotzer, 2012), whereas others suggest that it functions to inactivate the GTPase Rac1 (for example, Zhuravlev et al., 2017). Here, we review the mechanisms underlying RhoA activation during cytokinesis, primarily focusing on data in C. elegans. We highlight the importance of considering the parallel pathway for RhoA activation and detailed analyses of cyk-4 mutant phenotypes when evaluating the role of the GAP domain of CYK-4.

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