JCS00854 Supplemental Data

Files in this Data Supplement:

• Supplemental Figures 1,2,3 -

  Fig. S1. Double-labeling with GFP-tagged ER markers and an endogenous ER protein calnexin using a polyclonal antibody. Endogenous calnexin colocalized with GFP-KDEL (A), GFP-SERCA2a (B), and GFP-IP₃R1 (C). Images were taken with a confocal microscope. Scale bar, 10 µm.

  Fig. S2. Double-labeling with RFP-KDEL and (A) GFP-SERCA2a and (B) GFP-IP₃R1. GFP-tagged ER markers labeled both reticular ER and vesicles, while RFP-KDEL fluorescence was found only on vesicles. RFP-KDEL and GFP-tagged ER markers co-localized on the same vesicles (arrowheads). Images were taken with a confocal microscope. Scale bars, 10 µm.

  Fig. S3. (A) Expression pattern of GFP-LC3, a marker for autophagosomes, in a mouse hippocampal neuron. (B) Double labeling with RFP-KDEL and GFP-LC3. RFP-KDEL positive vesicles were distinct from GFP-LC3-labeled structure (circle). Images were taken with a confocal microscope. Bars indicate 10 µm.

• Movie 1
• Movie 2 -

  Movie 1 and Movie 2. Fluorescence of a part of the dendrite was bleached out by continuous laser illumination of 488 nm, 0.88 mW. During the photobleaching, images were taken every 50 ms under continuous illumination. Movie 1 shows the neuron expressing GFP-SERCA2a, and Movie 2 shows the neuron expressing GFP-IP₃R1. Illuminated areas are indicated by white circles. GFP signals on the reticular ER photobleached and never recovered during the illumination, while vesicular ER moved into the photobleached site of the dendrite and kept moving for ~15s. There is a shift in focus during the photobleach in movie 1, and this part was avoided from the analysis of vesicle movement (Fig. 2G).

• Movie 3 -

  Movie 3. Shows time-lapse images of dendrites of hippocampal neurons expressing RFP-KDEL. RFP-KDEL proteins were found only on particles. Vesicles labeled with RFP-KDEL were moving along the dendrites.