scholarly journals Drebrin-like protein regulates body bending of C. elegans via suppression of NCA cation leak channels

2019 ◽  
Author(s):  
Eugenia Butkevich ◽  
Peter Weist ◽  
Daniel Härtter ◽  
Dieter R. Klopfenstein ◽  
Renata Garces ◽  
...  
Keyword(s):  
Muscle Tone ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Force Microscopy ◽  
C Elegans ◽  
Guanine Nucleotide Exchange ◽  
Atomic Force ◽  
Nucleotide Exchange Factor ◽  
Genetic Epistasis

AbstractDrebrin-like protein (DBN-1) in C. elegans is an adaptor protein that connects different cellular pathways to the actin cytoskeleton. Using a CRISPR-Cas9 system, we generated a new dbn-1 allele, which lacks 80% of C-terminal part of DBN-1. The mutant displays a striking hyper-bending locomotion phenotype and body posture with two times stronger curvature than wild type. We show by atomic force microscopy that the muscle tone of the mutant remains unaffected. Aiming to track down the cause of hyper-bending, we performed genetic epistasis experiments. We found that mutations in the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio), pan-neuronal expression of dominant negative RHO-1 and mutations in NCA (NALCN) cation leak channels all suppressed hyper-bending in the dbn-1 mutant. These data indicate that DBN-1 negatively regulates the activity of both NCA-1 and NCA-2 channels, opposing RHO-1 in the non-canonical Gq pathway. We conclude that DBN-1 is an important component of the neuronal signaling cascade that controls the degree of C. elegans body bending during locomotion.

scholarly journals The NCA-1 and NCA-2 ion channels function downstream of Gq and Rho to regulate locomotion in C. elegans

2016 ◽  
Author(s):  
Irini Topalidou ◽  
Pin-An Chen ◽  
Kirsten Cooper ◽  
Shigeki Watanabe ◽  
Erik M. Jorgensen ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
C Elegans ◽  
Activating Mutations ◽  
Epistasis Analysis ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Forward Genetic Screens ◽  
Genetic Epistasis

AbstractThe heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans. Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans. Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

scholarly journals LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling

2017 ◽  
Vol 217 (1) ◽  
pp. 299-314 ◽  
Author(s):  
Hang Liu ◽  
Shimin Wang ◽  
Weijian Hang ◽  
Jinghu Gao ◽  
Wenjuan Zhang ◽  
...  
Keyword(s):  
Interacting Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Binding Partner ◽  
C Elegans ◽  
Intestinal Epithelia ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

RAB-10/Rab10 is a master regulator of endocytic recycling in epithelial cells. To better understand the regulation of RAB-10 activity, we sought to identify RAB-10(GDP)–interacting proteins. One novel RAB-10(GDP)–binding partner that we identified, LET-413, is the Caenorhabditis elegans homologue of Scrib/Erbin. Here, we focus on the mechanistic role of LET-413 in the regulation of RAB-10 within the C. elegans intestine. We show that LET-413 is a RAB-5 effector and colocalizes with RAB-10 on endosomes, and the overlap of LET-413 with RAB-10 is RAB-5 dependent. Notably, LET-413 enhances the interaction of DENN-4 with RAB-10(GDP) and promotes DENN-4 guanine nucleotide exchange factor activity toward RAB-10. Loss of LET-413 leads to cytosolic dispersion of the RAB-10 effectors TBC-2 and CNT-1. Finally, we demonstrate that the loss of RAB-10 or LET-413 results in abnormal overextensions of lateral membrane. Hence, our studies indicate that LET-413 is required for DENN-4–mediated RAB-10 activation, and the LET-413–assisted RAB-5 to RAB-10 cascade contributes to the integrity of C. elegans intestinal epithelia.

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.

scholarly journals Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

2008 ◽  
Vol 183 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Sophia Semerdjieva ◽  
Barry Shortt ◽  
Emma Maxwell ◽  
Sukhdeep Singh ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Steady State Levels ◽  
Endocytic Vesicles ◽  
Interfering Rna

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

scholarly journals REI-1 Is a Guanine Nucleotide Exchange Factor Regulating RAB-11 Localization and Function in C. elegans Embryos

2015 ◽  
Vol 35 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Aisa Sakaguchi ◽  
Miyuki Sato ◽  
Katsuya Sato ◽  
Keiko Gengyo-Ando ◽  
Tomohiro Yorimitsu ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
C Elegans ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Function

scholarly journals Regulation of type V adenylate cyclase by Ric8a, a guanine nucleotide exchange factor

2007 ◽  
Vol 406 (3) ◽  
pp. 383-388 ◽  
Author(s):  
Shyi-Chyi Wang ◽  
Hsing-Lin Lai ◽  
Yi-Ting Chiu ◽  
Ren Ou ◽  
Chuen-Lin Huang ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Guanine Nucleotide Exchange Factor ◽  
Dominant Negative ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
N Terminus ◽  
Type V

In the present study, we demonstrate that AC5 (type V adenylate cyclase) interacts with Ric8a through directly interacting at its N-terminus. Ric8a was shown to be a GEF (guanine nucleotide exchange factor) for several α subunits of heterotrimeric GTP binding proteins (Gα proteins) in vitro. Selective Gα targets of Ric8a have not yet been revealed in vivo. An interaction between AC5 and Ric8a was verified by pull-down assays, co-immunoprecipitation analyses, and co-localization in the brain. Expression of Ric8a selectively suppressed AC5 activity. Treating cells with pertussis toxin or expressing a dominant negative Gαi mutant abolished the suppressive effect of Ric8a, suggesting that interaction between the N-terminus of AC5 and a GEF (Ric8a) provides a novel pathway to fine-tune AC5 activity via a Gαi-mediated pathway.

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