GoKawiilDesign of the Cx34.7 and Cx35 mutant library
A semi-rational design approach was used to design the mutant library. Sequence alignments between the M. americana connexins and the connexins for which the most structure–function data existed (Cx26, Cx32, Cx36, Cx40 and Cx43) were performed in ClustalW. Sites identified by previous studies as conferring specificity for docking were used, as well as those identified by homology modelling from the structures of Cx26 (ref. 56). Specifically, we primarily focused on residues in the extracellular loops: four residues at the interface in EL2, KEVE/KDVE (M. americana Cx34.7 and Cx35), and one residue in EL1. The homologous residues in other connexins had been demonstrated to be highly tolerant to mutations and critical for docking specificity57. Mutations were modelled in Swiss PDB Viewer using homology models of Cx34.7 and Cx35 from a Cx26 and Cx32 interface structure so as not to create mutations with obvious steric hindrance. A wide range of substitutions were made for these five residues of interest, including those intended to introduce compatible electrostatic interactions, as well as less likely candidates. Mutations were also created that targeted other residues nearby and/or adjacent to these five for which there was some published evidence that they contributed to docking specificity. However, our semi-rational approach was such that not as many variants were evaluated for these more distal site mutations; mutations made in those sites were more conservative with regards to the steric and electrostatic properties of the change.
Construct cloning and preparation
The initially acquired M. americana Cx34.7 and Cx35 cDNA constructs did not express efficiently in HEK293FT cells. Thus, connexin gene information was procured from the National Center for Biotechnology Information (NCBI) and the Ensembl genome browser. The human codon-optimized genes were ordered from Integrated DNA Technology (IDT) as gBlocks Gene Fragments. To generate constructs for transient transfection of HEK293FT cells, genes were subcloned into BamHI-digested and SacI-digested mEmerald-N1 (Addgene, 53976) and piRFP670-N1 (Addgene, 45457) vectors using In-Fusion cloning (Takara Bio), which resulted in connexin fluorescent fusion proteins, specifically with the fluorescent proteins adjoined to the connexin carboxy terminus. Mutant constructs were generated by using overlapping primers in standard Phusion polymerase PCR reactions to facilitate site-directed mutagenesis.
The Gateway recombination (Invitrogen) system was used to generate all Cx36, Cx34.7, Cx35, WT and mutant protein C. elegans expression plasmids. For PCR-based cloning and subcloning of components into the Gateway system, either Phusion or Q5 High-Fidelity DNA polymerase (NEB) was used for amplification. All components were sequenced in the respective Gateway entry vector before combining components into expression plasmids via a four-component Gateway system58. The different connexin versions were introduced into pDONR221a using a similar PCR-based strategy from plasmid sources8,59,60. Cell-specific promoters were introduced using the pENTR 50-TOPO vector (Invitrogen) after amplification from genomic DNA or precursor plasmids. Transgenic lines were created by microinjection into the distal gonad syncytium61 and selected on the basis of expression of one or more co-injection markers: Punc-122::GFP or Pelt-7::mCherry::NLS.
Cell culture
HEK293FT cells were purchased from Thermo Fisher Scientific (R70007) and were maintained according to the manufacturer’s instructions. In brief, cultures were grown in 10-cm tissue culture-treated dishes in high-glucose DMEM (Sigma Aldrich, D5796) supplemented with 6 mM l-glutamine, 0.1 mM MEM non-essential amino acids and 1 mM MEM sodium pyruvate in a 5% CO 2 , 37 °C incubator. Cells were passaged by trypsinization every 2–3 days or until 60–80% confluency was reached. The cell line identity was not validated beyond the manufacturer’s certification of authenticity, and it was not tested for mycoplasma contamination. HEK293FT cells are not included in the list of commonly misidentified cell lines published by the International Cell Line Authentication Committee.
Transient transfection
HEK293FT cells were plated in 10 μg ml–1 fibronectin-coated multiwell dishes to achieve about 75% confluency after overnight incubation. For transfection, 250 ng DNA was combined with polyethylenimine (PEI) diluted in Opti-MEM in a 1:3 ratio (DNA (µg) to PEI (µl)) and incubated at room temperature for 10 min. Following incubation, PEI–DNA complexes were added dropwise to wells of the plated cells. Treated cells were then incubated at 37 °C for 16–18 h, followed by a change in the medium. Expression of the connexin–fluorescent protein constructs were evaluated at 24 and 48 h after transfection by widefield or confocal microscopy and western blotting.
FETCH
... continue reading