Description

UNC-11 is an ortholog of mammalian PICALM (phosphatidylinositol binding clathrin assembly protein) and its neuron-specific paralog, AP180 (Maritzen et al. 2012). PICALM facilitates recycling of Vesicle Associated Membrane Proteins (VAMPs) from the plasma membrane (Miller et al. 2011, Sahlender et al. 2013 and Miller et al. 2015) and efficient clathrin-coated endocytosis (Huang et al. 2004, Miller et al. 2015 and Ishikawa et al. 2014). unc-11 function in C. elegans is likely conserved as SNB-2 (VAMP2) and the glutamate receptor, GLR-1, both accumulate at the plasma membrane in unc-11(e47) null mutants (Nonet et al. 1999, Burbea et al. 2002). Mutant phenotypes outside the worm nervous system have not been described.

To determine the endogenous expression pattern and subcellular localization of unc-11, we created a functional GFP knock-in at the endogenous unc-11 locus using the CRIPSR approach outlined by Dickinson et al. 2015. Specifically, a GFP::AID::3xFLAG coding sequence interrupted by four synthetic introns was inserted just 5′ to the unc-11 stop codon. Successful knock-in was verified by sequencing the insertion junctions and expression of GFP.

To ask if the addition of GFP at the C-terminus of UNC-11 compromises unc-11 function we performed a thrashing assay to examine the general health of the nervous system (Figure 1B). We compared the thrash duration (see methods) of unc-11(+) with the CRISPR knock-in strain, unc-11(vg103) and the null mutant, unc-11(e47). We found no obvious differences between unc-11(+) and unc-11(vg103). Average thrash duration during a 45 second interval was 0.251 seconds (n=9, Std. Dev. = 0.042) for unc-11(+) and 0.248 seconds (n=9, Std. Dev. = 0.031) for unc-11(vg103). By contrast, the average thrash duration for unc-11(e47) was significantly higher at 0.566 seconds with a striking increase in variability (n=9, Std. Dev. = 0.196).

In general, UNC-11::GFP expression matched that of UNC-11 antibody staining described previously (Nonet et al. 1999) with some noted exceptions. Similar to UNC-11 antibody staining, UNC-11::GFP expression appeared pan-neuronal and was also observed within coelomocytes (Figure 1A and 1D). Not described by Nonet el al 1999, we observed GFP puncta in apical epithelial tissue just below the cuticle surface in both the head and in the body (Figure 1E and 1F), an accumulation of UNC-11::GFP at the plasma membrane in the seam cells (Figure 1F) and weak staining in the vulval precursor cells (VPCs) at the interface between the VPCs and the vulval lumen (Figure 1C). Finally, Nonet et al. described diffuse staining within the intestine whereas we did not observe detectable levels of intestinal expression, punctate or otherwise (Figure 1A).

Plasmid construction was done in the context of a course entitled Advanced Molecular Techniques at California State University, East Bay. This strain will be made available at the CGC. Given the inclusion of an AID tag and GFP, this strain will be useful for cell-specific protein degradation using auxin-inducible (AID) or GFP nanobody::ZIF-1 degradation methods (Wang et al. 2007 and Zhang et al. 2015).