Figure 9.

UNC-69 and UNC-76 colocalize as puncta in neuronal processes. (a-o) Functional P_unc-69::cfp::unc-69 and P_unc-76::unc-76::yfp constructs were coinjected at 5 ng/μl each into unc-76(e911) mutants, and worms rescued for locomotion were selected. Note that the unc-76(e911) mutation was removed from the background in (g-o). (d-o) are deconvoluted single-layer images. (a-c) Lateral view of an adult hermaphrodite from one line of transgenic animals with a wild-type phenotype. Both CFP::UNC-69 and UNC-76::YFP form discrete, large puncta in the DNC, as well as in the commissure (arrow). Vignette in (c) shows an enlarged image of colocalized puncta in the DNC from the rectangle. (d-f) Lateral view of an adult hermaphrodite from a second line of transgenic animals with a wild-type phenotype. Note that CFP::UNC-69 and UNC-76::YFP are both cytoplasmic and punctate, and the puncta are present in lateral and sublateral processes. (g-i) In unc-116(rh24) mutants, UNC-76::YFP puncta became diffuse in a stretch of axon in the VNC, and failed to colocalize with CFP::UNC-69 (arrows in (h,i)). (j-l) CFP::UNC-69 and UNC-76::YFP colocalize in perinuclear structures in the soma of a neuron in the tail ganglia. (m-o) In unc-116(rh24) mutants, both UNC-76::YFP and CFP::UNC-69 often appear as partially overlapping or non-overlapping aggregates in the soma of a (i) preanal and (ii-iii) two tail ganglion neurons. (p-u) Expression pattern of (p-r) opIs124 (P_unc-69::unc-69::gfp) and (s-u) opIs130 (P_unc-76::unc-76::yfp). Both transgenes were integrated into the genome to ensure stable gene expression. All pictures show the CAN neuron soma (arrowhead) and its vicinity. (p,s) In wild-type worms, the CAN neuron extended its bipolar processes along the excretory canal, and the CAN neurites were filled with UNC-69::GFP and UNC-76::YFP. Note that puncta cannot be seen in these integrants owing to overexpression of the transgenes. (q) In unc-104(e1265) mutants, UNC-69::GFP accumulated near the CAN soma as well as in its neuronal processes (asterisks), giving it a notched appearance. (t) UNC-76::YFP localization appeared to be grossly normal in unc-104(e1265) mutants. (r,u) In unc-116(rh24) mutants, both UNC-69::GFP and UNC-76::YFP accumulated in CAN neurites (asterisk). UNC-69::GFP accumulation was prominent near the CAN soma and was accompanied by ectopic branches. In contrast, UNC-76::YFP aggregated and was evenly distributed along the CAN processes. The scale bar represents 20 μm. (v,w) The CAN neuron visualized by the integrated transgene kyIs4 (P_ceh-23::ceh-23::unc-76_1–197::gfp). (v) In wild-type worms, GFP appeared as string of dots, reminiscent of endogenous UNC-76 expression pattern. (w) In unc-116(rh24) mutants, GFP dots became larger and more dispersed. (x,y) CAN neuron visualized by an extrachromosomal array opEx901 (P_unc-69::gfp). Unlike the UNC-69::GFP fusion (r), GFP itself did not accumulate significantly around CAN soma in unc-116(rh24) mutants (y), although ectopic branches were frequently observed. (p-y) are confocal z-stack images. Anterior is to the left in (a-i) and (p-y); anterior is up and ventral is to the right in (j-o). All scale bars except in (p-u) represent 10 μm.