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. Author manuscript; available in PMC: 2019 Aug 11.
Published in final edited form as: J Neurosci Methods. 2018 May 21;306:94–102. doi: 10.1016/j.jneumeth.2018.05.014

Figure 5.

Figure 5.

Assessment of trans vs cis GFP reconstitution at the third instar larval NMJ. A-E) vGlut-LexA drives expression of pre-t-GRASP in motor neurons and 24B-GAL4 drives expression of post-t-GRASP in muscles. F-J) 24B-GAL4 drives expression of pre-t-GRASP in muscle and vGlut-LexA drives expression of post-t-GRASP in motor neurons. K-O) vGlut-LexA drives simultaneous expression of both pre-t-GRASP and post-t-GRASP in motor neurons. GRASP signal (green) is observed when pre-t-GRASP (red) is expressed in motor neurons and post-t-GRASP (cyan) is expressed in muscle (arrow A), but not when post-t-GRASP is expressed in motor neurons and pre-t-GRASP is expressed in muscle (F) or when both pre-t-GRASP and post-t-GRASP are expressed in motor neurons (K). pre-t-GRASP accumulates in the pre-synaptic terminals of motor neurons (arrows B, L) and at the post-synaptic region of muscles (G). post-t-GRASP accumulates outside the nucleus and at the post-synaptic region of muscles (C) and in the pre-synaptic terminals of motor neurons (arrows H, M). Overlay of GRASP signal with pre-t-GRASP (D, I, N) and with pre-t-GRASP and post-t-GRASP (E, J, O). The observation that GRASP signal is only observed when pre-t-GRASP is expressed in motor neurons and post-t-GRASP is expressed in muscles, but not in the reciprocal experiment or when both pre-t-GRASP and post-t-GRASP is expressed in motor neurons reveals the t-GRASP method exhibits a significant preference for extracellular trans over intracellular cis GFP reconstitution. Reconstituted GFP, pre-t-GRASP, and post-t-GRASP were visualized with anti-GFP Tag Abfinity mAb, anti-HA, and anti-TLN antibodies, respectively. Scale bar 30μm.

Figure 5 genotypes. A-E) yw; vGlut-LexA/+ ;24B-GAL4/13XLexAop2-pre-t-GRASP, 20XUAS-post-t-GRASP; F-J) yw; vGlut-LexA/+; 24B-GAL4/20XUAS-pre-t-GRASP/13XLexAop2-post-t-GRASP; K-O) yw; vGlut-LexA/+; 13XLexAop2-pre-t-GRASP, 20XUAS-post-t-GRASP.