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 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 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 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 An Arf/Rab cascade controls the growth and invasiveness of glioblastoma

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Gopinath Kulasekaran ◽  
Mathilde Chaineau ◽  
Valerio Emilio Crescenzo Piscopo ◽  
Federica Verginelli ◽  
Maryam Fotouhi ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Tumor Initiating Cells ◽  
Human Brain Tumor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Brain Tumor Initiating Cells

Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

scholarly journals PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

2019 ◽  
Author(s):  
Sophie Vieweg ◽  
Katie Mulholland ◽  
Bastian Bräuning ◽  
Nitin Kachariya ◽  
Yu-Chiang Lai ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Therapeutic Potential ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Lrrk2 Kinase ◽  
Genetic Code Expansion

AbstractLoss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

scholarly journals An Arf/Rab cascade controls the growth and invasiveness of glioblastoma

2020 ◽  
Author(s):  
Gopinath Kulasekaran ◽  
Mathilde Chaineau ◽  
Valerio E. Piscopo ◽  
Federica Verginelli ◽  
Maryam Fotouhi ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Tumor Initiating Cells ◽  
Human Brain Tumor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Brain Tumor Initiating Cells

AbstractGlioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brain. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn and allosterically enhances its GEF activity towards Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals upregulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

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