The neural G protein Gαo tagged with GFP at an internal loop is functional in Caenorhabditis elegans

Abstract

Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.

Dynein directs prophase centrosome migration to control the stem cell division axis in the developingC. elegansepidermis

“…Work in the early embryo identified the Gαi subunits GOA-1 and GPA-16 as the plasma membrane receptors for GPR-1/2, which in turn recruit LIN-5 (Colombo et al ., 2003; Gotta et al ., 2003; Srinivasan et al ., 2003). To determine whether this pathway operates in seam cells, we first asked whether GOA-1 is present in seam cells using a transgene that expresses functional GOA-1, internally tagged with GFP, from goa-1 regulatory elements (Kumar et al ., 2021). Co-expression with mCh::HIS-11 and mCh::PH showed that GOA-1::GFP localizes uniformly to the seam cell plasma membrane at all stages of the cell cycle, which contrasts with the fluctuating levels and bipolar distribution of LIN-5::mNG ( Fig.…”

“…To the best of our knowledge, Gαi localization in mitosis has not been examined by live cell imaging in any organism. We took advantage of a recently generated functional GFP-tagged version of GOA-1 (Kumar et al ., 2021) to examine GOA-1 localization in seam cells. This revealed that GOA-1 is uniformly distributed on the cortex throughout mitosis, which contrasts with the fluctuating levels and uneven distribution of LIN-5 NuMA .…”

The G protein alpha chaperone and guanine-nucleotide exchange factor RIC-8 regulates cilia morphogenesis in Caenorhabditis elegans sensory neurons

“…2 p) [ 76 ] to ensure that results are not confounded by BiFC complex formation during development. Additionally, we expressed full-length ODR-3 tagged in an internal loop of its alpha-helical domain (see Materials and Methods ) with the N-terminal Venus fragment (ODR-3 FL ::VN) [ 77 ] in AWC neurons of the same animals. Importantly, we demonstrate that the internal tag does not interfere with ODR-3 function, as the internally tagged ODR-3 FL ::TagRFP fusion protein localizes to cilia when expressed in AWC (see Fig 5A ) and rescues odr-3 -dependent cilia defects ( S2 Fig ).…”

“…Since formation of the bimolecular fluorescent complex can be irreversible [75], we expressed full-length RIC-8 tagged with the Venus C-terminal fragment (RIC-8 FL ::VC) in wild-type animals under a heatshock promoter (hsp-16.2p) [76] to ensure that results are not confounded by BiFC complex formation during development. Additionally, we expressed full-length ODR-3 tagged in an internal loop of its alpha-helical domain (see Materials and Methods) with the N-terminal Venus fragment (ODR-3 FL ::VN) [77] in AWC neurons of the same animals. Importantly, we…”

“…TagRFP-tagged odr-3 constructs: odr-3 coding sequences corresponding to amino acids (AA) 1-118 and AA 119-356 were amplified from a mixed-stage N2 cDNA library using Phusion DNA polymerase (NEB) with gene-specific primers. Tagrfp cDNA with flanking DNA segments encoding SGGGGS linkers was amplified using Phusion DNA polymerase (NEB) and inserted between Gly118-and Glu119-encoding codons of odr-3 [77] in the modified PLOS GENETICS pMC10 vector using NEBuilder HiFi DNA assembly (NEB). The resulting plasmid sequence was confirmed by Sanger sequencing.…”