Figure 3. Epitope-tagged MCTP localizes to ER membranes.
(A) Image shows the distribution of epitope-tagged MCTP in a single bilaterally symmetrical pair of motoneurons in the larval CNS (MN1-Gal4>UAS-mctp-Flag, green) The dotted line indicates the CNS midline and the numbers refer to inset images at right showing single confocal sections of peri-nuclear reticular membranes in the soma (1, 2) and the MCTP-labeled membranes that continue into the axon and dendrites (3). (B) Synaptic distribution of HDEL-GFP (green, top), imaged in a live preparation. Below, MCTP-Flag (red) is shown for the same NMJ following mild fixation and antibody labeling. The yellow box indicates the region shown at higher magnification to the right. The live HDEL-GFP and post-fixation MCTP-Flag identify the same tubular organization of the ER membranes within an individual synaptic bouton. No staining on the neuronal plasma membrane is evident. The NMJ is co-stained with a marker of the neuronal plasma membrane (anti-HRP, blue). Scale bar: 10 µm. (C) Structured illumination microscopy (SIM), single confocal section images of a synaptic bouton expressing the active zone marker Brp-GFP (driven by its endogenous promoter, green) and MCTP (OK371-Gal4>UAS-MCTP-Flag, red). Arrows indicate regions where MCTP resides in close proximity to Brp puncta, which define the T-bar structures that reside at the center of individual active zones. Scale bar: 1 µm. (D) Axon membranes from the peripheral nerve stained as in (B). Scale bar: 50 µm. (E) Single confocal sections of individual S2 cells transfected with MCTP-GFP and co-labeled with ER-tracker (red; see Materials and methods). At left, an S2 cell transfected with full length MCTP-GFP. At middle, an S2 cell transfected with a transgene expressing only the two-transmembrane region of MCTP linked to GFP (TM only-GFP). ER localization is retained. At right, an S2 cell transfected with a transgene expressing an MCTP-GFP transgene lacking the two-transmembrane domain and ER localization is lost (MCTP-noTM-GFP). Scale bar: 10 µm.