Figure 2

Transmission of Ca²⁺ signals and membrane constituents along membrane nanotubes. (a) Nanotubes between two MDA-MB-231PA cells visualized using widefield fluorescence microscopy by a deep red plasma membrane stain. (b) The same two cells showing fluorescence of the Ca²⁺ indicator Cal-520 (F, arbitrary units) before and after UV laser spot photorelease of i-IP₃ delivered at t = 22 s at the location marked by the asterisk in the first panel. White lines and numbers indicate kymograph section and annotations used to generate panel d. (c) Traces show Ca²⁺ fluorescence signals evoked from 7 representative cells directly stimulated at 22 s by photorelease of i-IP₃ (red), and signals in responding, nanotube-connected cells (black). (d) Kymograph image (with time depicted vertically and distance horizontally) formed by measuring Ca²⁺ fluorescence changes (ΔF/F₀) between the lines marked white in (b) as a function of time following UV spot stimulation of the upper cell. Notations (1, 2, 3, 4) mark corresponding locations in the cell image and kymograph to delineate sites within each cell and the nanotube connecting them. Arrow marks the time of the UV flash (e) Snapshot images captured via lattice light-sheet microscopy illustrating movement of a GFP-tagged membrane constituent along the length of the nanotube at three times as indicated. Arrows mark the locations of the vesicle. Scale bar, 2 µm. See also Movie 2. (f) Distance-time plots of membrane constituents moving along nanotubes. Inset graph shows a distribution histogram of mean track velocities. Data are from 22 representative nanotubes in 3 separate cultures.