rab-33 is not required for cuticle integrity in Caenorhabditis elegans

Abstract

Rab GTPases are master regulators of intracellular transport. We previously showed that a rab-6.2 deletion leads to a compromised cuticle, glycosylation defects, and increased axon regeneration. Mammalian orthologs of rab-6.2 and rab-33 have been shown to mediate Golgi trafficking of cargo in mammalian cells, including that of glycosyltransferase enzymes. We engineered a novel STOP-IN putative null allele of rab-33 ( axr2 ) to determine if loss of function of rab-33 phenocopies the phenotypes seen in rab-6.2 ( ok2254 ). Our results suggest that rab-33 is not required for cuticle integrity.

Figure 1. CRISPR Design and Cuticle Integrity Characterization of rab-33 mutants

A) Diagram showing the rab-33 and taf-11 .3 loci and the ok1561 deletion, dashed lines represent additional sequence upstream of the rab-33 locus. The diagram also shows the genetic construct (STOP-IN cassette, adapted from Wang et al. 2018) used in this study and the CRISPR insertion site in the rab-33 gene; HDR arms are highlighted (green) on both ends of the construct, Synthetic Nhe-1 restriction enzyme site (purple), early stop codon is noted by an asterisk, external and internal genotyping primers are shown. Lower panel shows sanger sequencing data using a rev primer, confirming the correct insertion of the STOP-IN cassette in rab-33 ( axr2 ) homozygotes. B) Genotyping results confirming correct insertion of STOP-IN cassette in our mutants. The external amplicon is also digested with NheI to confirm insertion. C) Results of 150 μM Tetramisole paralysis assays on day-1 adult N2 worms (wild type), rab-6.2 ( ok2254 ) worms, rab-33 taf11-.3 ( ok1561 ) and the newly engineered rab-33 ( axr2 ) . P-value < 0.0001, Log-rank (Mantel-Cox) test. D) Results of 125 μM Levamisole paralysis assays after 30 minutes on day 1 adult N2 worms, dpy-10 ( cn64 ) worms, and rab-33 ( axr2 ). Error bars denote standard deviation. P-value < 0.001. E) images of heads and tails of N2 , dpy-10 ( cn64 ) , and rab-33 ( axr2 ) D1 adult worms stained with Hoescht 33258. Arrowheads show stained nuclei in the dpy-10 ( cn64 ) worms, which both the N2 and rab-33 ( axr2 ) do not exhibit. F) percentage of animals displaying nuclear stains in heads or tails. Scale bar = 20mm. Error bars denote standard deviation. p < 0.001.