Properties of NG2 cell processes.
(A–D) Two-photon time-lapse recordings. (A) Overview of an Alexa-594-labeled NG2/EYFP cell (maximum projection, 100 µm x 100 µm x 15 µm, 60 equidistant planes, scale 10 µm). (B–D) Pairs of maximum projections (16 µm x 14 µm x 5 µm, 20 planes, scale 2 µm) taken at time points t0 (left) and t0 + Δt (right). Arrows mark processes that were elongated (B, Δt = 185 s) or retracted (C, Δt = 370 s). Additionally we observed varicosities traveling along the process (D, Δt = 370 s, start and end point marked by arrows). See also Videos S2, S3. (E–H) NG2 cell processes do not contain α-tubulin and PDI. Cortical tissue from an hGFAP/EGFP mouse (p13) was freshly dissociated and quadruple-stained with a nuclear marker (bisbenzimidine, blue) and antibodies against GFAP (also blue channel), GFP (green) and one of the proteins of interest (red): α-tubulin (E), β-actin (F), ezrin (G) or the ER marker PDI (H). The cells analyzed were GFAP negative, GFP positive. Note nearby GFP negative cells (overviews, left in F–H, E). Areas boxed in the overviews (F–H) are enlarged for colocalization analysis. β-actin and ezrin were localized in the NG2 cell processes. Note the fine dimensions of these varicose processes visualized in the GFP channel (green). The PDI signal is present both in a non-identified, nearby cell (H, overview) and in the soma of the NG2 cell, but not in its processes (H, red, merge). The same is observed for α-tubulin (E red, merge). Note that α-tubulin/microtubules are well-preserved in the processes of nearby non-identified cells (E, merge) and of GFAP positive astrocytes (cf. Fig. S1). Scale bar 5 µm.
