Fig. 1. ATP depletion affects ER mobility without altering luminal crowdedness.

(a) Trace of time-dependent decay in the intensity of the fluorescence signal from an ER-localised photoconvertible protein, Dendra2-ER, after a pulse of photo-converting illumination delivered to a small patch of untreated or ATP-depleted COS7 cells. Inset denotes mean ± SEM, (n=5 traces per condition) of fluorescence decay half time, reflecting the probe’s escape from the photoconversion area.

(b) Fluorescence intensity (left) and colour-coded fluorescence lifetime (FLT) images (right) of COS7 cells transfected with an ER-localised molecular crowding probe⁷. FLT distribution within imaged cells is displayed in colour-coded histograms with mean FLT noted (in picoseconds, ps, right). Cells were left untreated (UNT), ATP depleted, or treated with hyper-osmotic (Hyper Os.) or hypo-osmotic buffers that induce cell shrinking or swelling to obtain maximal and minimal crowding values, respectively. Shown are characteristic images observed in three independent repeats.

(c) Bar diagram of FRET-donor FLT values measured as in (b) (mean values ± SD, n=22 independently sampled cells).

(d) Bar diagram of relative intracellular ATP concentration measured with FRET-based ATP-probe (A-Team)²³ in cells untreated or ATP depleted as in (a & b). Minimum and maximum values represent the probe readings in ATP depleted or saturating conditions respectively, imposed in semi-permeabilised cells. Shown are mean values ± SD, n=10 independently sampled cells.

(e) Images of COS7 cell expressing Dendra2. A brief pulse of illumination photoconverted Dendra2 from green to red in a restricted region of the ER. The progression of the photoconverted packet of proteins is revealed by the time series and summated in the bottom panel with its velocity colour coded.