Figure 4

unc-2 mutants exhibit fewer dynamic neuromuscular junctions (NMJs) during the 4th larval (L4) stage. SNB-1::GFP puncta formed in the ventral cord of wild type or unc-2(e55) animals were imaged for one hour. The panels are a mask of the threshold image and false colored to demonstrate changes in the shape of the puncta. In wild type after one hour the puncta largely align (yellow), with some differences, highlighted in the boxed regions. (A-C1) Three small puncta (arrowheads) that are present at time 0 have disappeared 60 minutes later (B1). The elongated punctum (arrow) has condensed. (A-C2) An elongated punctum present at time 0 (arrow) appears to have separated into two distinct puncta. An adjacent punctum (arrowhead) is largely unchanged. (A-C3) An empty space at time 0 has two puncta (arrows) that have formed. (D-F) In unc-2 mutants puncta generally enlarged, but we observed fewer formation/elimination and division events. (D-F1) One punctum (arrow) was approximately twice as large after 60 minutes, while the adjacent puncta were largely unchanged. Note: the animal moved slightly during the protocol, putting the puncta in D1-F1 slightly out of alignment. (D-F2) We found occasional puncta that appeared to be elongated (arrowhead). In unc-2 these puncta remained elongated after an hour whereas in wild type they often resolved into two puncta, or shrunk in size. (A-C2). (D-F3) Many SNB-1::GFP puncta were approximately the same size, undergoing little change during the time lapse, indicating that SNB-1::GFP was not affected by photobleaching over the imaging protocol.