PMC

FM4-64 uptake involves a SOCE pathway. FM4-64 labeling (6.7 μM, 5 min) was increased by TG treatment (2 μM, 30 min) (A), and calyculin A (Caly, 200 nM, 60 min) (B), and inhibited by anisomycin (Aniso, 30 μM, 12 h) (C), Gd³⁺ (100 μM, 10 min) (E), BTP2 (40 μM, 10 min) (D), and SKF96365 (20 μM, 10 min) (F).

FM dyes uptake in astrocytes involves a cationic channel. (A) Epifluorescence images of astrocytes labeled with either FM4-64 or its spectral analogue FM5-95 (6.7 μM, 5 min each). Loading is enhanced with the smaller FM5-95. (B) Similar labeling to FM4-64 is seen with short lipophilic tail FM1-43 (12 μM), but FM3-25 (20 μM), having a longer lipophilic tail, is virtually impermeant. Quenching extracellular FM1-43 and FM3-25 with 2 mM BPB permitted quantification of the intracellular labeling. (C) Incubation of the astrocytes in high [K⁺]_o solutions reduced FM4-64 uptake. (D) Incubation of the astrocytes with FM4-64 at high [Ca²⁺]_o (20 mM, h-Ca) diminished FM dye uptake compared with control (1.8 mM) medium [control (CTR); n = 7–25 cells per condition]. (Scale bars: 10 μm.)

FM dye insertion in the PM outer lipid leaflet activates a SOCE. (A) Preincubating astrocytes with GsMTx-4 (5 μM, 10 min) reduced the FM4-64 uptake (6.7 μM, 5 min). (B) Treating astrocytes with LPC (5 μM, 10 min) increased the FM4-64 uptake. Arachidonic acid (AA, 10 μM, 10 min) had no effect. (C) Treating astrocytes with MβCD (10 mM, 1 h) diminished FM4-64 labeling. (D) The local application of green impermeant FM3-25 facilitated SOCE. The ER store was depleted by treating cells with TG (1 μM, 10 min) in Ca²⁺-free buffer. The [Ca²⁺]_i was monitored with the red Ca²⁺ indicator Xrhod-1 (n = 7–16 cells per condition). (Scale bars: 10 μm.)

Endocytosis is not a major route for rapid FM dye uptake in cultured astrocytes. (A) Epifluorescence image of a subregion of a cortical astrocyte loaded with FM4-64 (6.7 μM, 2 min incubation at RT, 10-min wash). (Scale bar, 5 μm.) Traces (gray, individuals; color, mean ± SD) display the evolution with time of the fluorescence intensity upon membrane rupture, measured in the extracellular (turquoise, 11 ROIs from 5 cells), cytoplasmic (red; 13 ROIs), and vesicular ROIs after local background subtraction (blue, 14 ROIs). (B) Top: Pearson correlation coefficient, calculated for subcellular ROIs between FM dyes and endocytic markers, coloaded for 5 min (Fig. S1B in SI Appendix). Bottom: Labeled puncta density (per μm²). (C) TIRFM images of astrocytes loaded with FM4-64 (6.7 μM, 5 min) at RT (control; CTR) and 4 °C (T_chamber, 3.7 ± 0.4 °C, n = 25). (Scale bar: 10 μm.) (D) FM4-64 labeling in control and after pretreatment with brefeldin A (Bfa, 5 μg/mL, 30 min) or dynasore (Dyn, 50 μM, 30 min; P = 0.2–0.77, n = 8–15 cells per condition). (E) FM4-64 labeling in control and after pretreatment with cytochalasin D (Cyt, 5 μM, 60 min) or jasplakinolide (Jasp, 2 μM, 60 min; P = 0.16–0.69, n = 5–18 cells). *, P < 0.05; **, P < 0.01.

Permeant FM dyes modifies Ca²⁺ signaling in cultured astrocytes. (A) After depletion of ER Ca²⁺ store with TG in 0 mM Ca²⁺, an SOCE was induced by local application of control (CTR) solution (1.8 mM Ca²⁺, Upper) or by CTR solution containing 6.7 μM FM4-64 (Lower). Monitoring [Ca²⁺]_i with the green Ca²⁺ indicator OGB-1, we show that FM4-64 reduces the SOCE (mean ± SD, black and gray traces). (B) We locally applied control solution (1.8 mM Ca²⁺; CTR) first, followed by the application of FM4-64-containing solution (6.7 μM FM4-64 and 1.8 mM Ca²⁺; FM), before returning to the control. FM4-64 evokes a [Ca²⁺]_i elevation (green trace) that follows the FM dye insertion inside the outer PM lipid leaflet (red trace), measured with TIRFM. Inset: Same traces magnified to show the onset of the response to FM4-64. (C) Gd³⁺ (100 μM) and GsMTx-4 (2.5 μM) both reduced FM4-64 uptake (ΔF_FM measured at the end of the 2 min FM4-64 wash, B) and the FM-triggered [Ca²⁺]_i transient (dF/F₀ measured at the peak of the Ca²⁺ response, B). Removing extracellular Ca²⁺ (0 Ca²⁺, 5 mM EGTA) facilitated both the FM4-64 uptake and the Ca²⁺ response. (D) Pretreatment of astrocytes with TG (2 μM, 20 min) enhanced FM4-64 uptake and suppressed the FM dye-induced mobilization of intracellular Ca²⁺ store. (E) In astrocytes transfected with an ER-targeted esterase, local application of FM4-64 (6.7 μM) induced a reduction of the ER Ca²⁺ concentration ([Ca²⁺]_ER) monitored with the low-affinity Ca²⁺ indicator fluo5N-AM. Top: Dual-color Fluo5N and FM4-64 TIRFM kymographs. Bottom: FM4-64 insertion into the PM (red trace) was associated with a reduction of [Ca²⁺]_ER (green trace; n = 5–10 cells per condition).