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. 2019 Apr 24;13:164. doi: 10.3389/fncel.2019.00164

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Copyright © 2019 Lenoir, Varangot, Lebouvier, Galli, Hommet and Vivien.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Dendritic and axonal release of tPA. (A) Timeline of the experiment. (B) Schematic diagrams of a dual color reporter for neuronal tPA. tPA fused to a HaloTag® site and the superecliptic pHluorin (SEP) allows simultaneous visualization of total (HaloTag®) and extraneuronal (SEP) tPA molecules. (C) Representative z-stack confocal images of transfected cortical neurons (DIV12) with pCMV_HaloTag®-tPA-SEP plasmid (TMR ligand; in red and SEP; in green), time-lapse were acquired in 2 dimensions every 30 s (t = 0 was assigned of exocytosis; Time is in min:sec). After live imaging ICCs were performed to reveal MAP2 (in blue) and thus to differentiate axons from dendrites (an example of each are shown). Respective kymographs of HaloTag®-tPA-SEP exocytosis are shown. Scale bar represents 2 μm. (D) Histograms show the percentages of exocytotic tPA-containing vesicles (SEP positives puncta, in green) compared to the total pool of tPA-containing vesicles (TMR positives puncta, in red) in axons (9%) and in dendrites (19%) (N = 16 and N = 14 neurons respectively, from N = 7 independent cultures, p = 0.007). (E) Schematic histograms of the number of vesicles exocytosed per 100μm in axons (6) and dendrites (11) (N = 13 and N = 11 neurons respectively, from N = 5 independent cultures, p = 0.034).