Agonist-induced regulation of mitochondrial and endoplasmic reticulum motility

Abstract

Using fluorescently tagged markers for organelles in conjunction with confocal microscopy, we have studied the effects of agonist-induced Ca2+ signals on the motility of mitochondria and the ER (endoplasmic reticulum). We observed that the muscarinic agonist carbachol produced a rapid, simultaneous and reversible cessation of the movements of both organelles, which was dependent on a rise in cytosolic Ca2+. This rise in Ca2+ was shown to cause a fall in cellular ATP levels, and the effect of carbachol on organelle movement could be mimicked by depleting ATP with metabolic inhibitors in the absence of any such rise in Ca2+. However, a Ca2+-sensing process independent of ATP appears also to be involved, because we identified conditions where the ATP depletion was blocked (by inhibitors of the Ca2+ pumps), but the organelle movements still ceased following a rise in cytosolic Ca2+. We conclude that the co-ordinated cessation of mitochondria and ER motility is a process regulated by the cytosolic concentration of both Ca2+ and ATP, and that these two parameters are likely to synergize to regulate the localization of the two organelles, and to facilitate the transfer of Ca2+ between them.

Figure 1. The effects of 100 μM carbachol (CCh) on organelle mobility

(A) HEK-293 cells stably expressing GFP targeted to the ER (i) and loaded with Mitotracker Red (ii) illustrate the respective organelle morphology and distribution. Scale bar represents a distance of 10 μm. (B) The change in organelle mobility, described here as the MI, following the addition of CCh for both the ER (continuous line) and mitochondria (Mito; broken line). See Supplementary Movie 1 (http://www.BiochemJ.org/bj/392/bj3920291add.htm for a full version of this Figure.

Figure 2. Ca²⁺-dependence of inhibited organelle movement

(A) Correlation of [Ca²⁺]_i with ER mobility in response to CCh. The change in [Ca²⁺]_i is recorded as a change in Fura Red fluorescence (broken line), and is expressed in arbitrary units. Effects on ER mobility (continuous line) are expressed as the change in MI. (B) The change in ER (continuous line) and mitochondrial (Mito; broken line) mobility in response to CCh, and addition of Ca²⁺ to the bathing solution under conditions of low extracellular Ca²⁺, followed by re-addition of Ca²⁺. (C) The effects of CCh on ER (continuous line) and mitochondrial (broken line) mobility in cells pre-incubated with the Ca²⁺-chelator BAPTA/AM under conditions of normal extracellular Ca²⁺.

Figure 3. Dependence of organelle movement on [ATP]

(A) The effects of interventions (combinations of 2-DOG and CCCP) designed to have a transient (i, ii) or permanent (iii, iv) effect on cellular [ATP] in relation to ER (continuous line) and mitochondrial (Mito; broken line) mobility. (B) Effects of CCh and metabolic inhibitors on [ATP]. HEK-293 cells transiently expressing cytosol-targeted firefly luciferase, bathed in D-luciferin, stimulated with CCh followed by an intervention designed to permanently reduce cellular [ATP] (see above and the main text for further details). Bioluminescence is expressed as counts/second (CPS).

Figure 4. Effects of ATP depletion on [Ca²⁺]_i

(A) WT HEK-293 cells were loaded with Fluo-4/AM and stimulated with either 100 μM CCh or 1 μM thapsigargin (Tg). The black trace represents the mean CCh response (four replicates), and the grey trace represents the mean thapsigargin response (four replicates). The data are presented as F/F_o. (B) WT HEK-293 cells were loaded with Fluo-4/AM and stimulated with interventions (combinations of 2-DOG and CCCP) designed to have transient or permanent effects on cellular [ATP] (cf. Figure 3). The key featured as an inset indicates the treatments applied. The data represent the means of four replicates, and are shown as F/F_o.

Figure 5. Organelle movement, [ATP] and [Ca²⁺]_i in cells incubated with 1 mM Gd³⁺

The effects of 100 μM CCh (black trace) and 1 μM thapsigargin (Tg; grey trace) on [Ca²⁺]_i in WT HEK-293 cells loaded with Fluo-4/AM. Data are the means of nine replicates, and are represented as F/F_o. (B) Effects of Tg on [ATP]. HEK-293 cells transiently expressing cytosol-targeted firefly luciferase were bathed in D-luciferin and stimulated with Tg, followed by an intervention designed to permanently reduce cellular [ATP]. Bioluminescence is expressed as counts/second (CPS). (C) The mobility of the ER and the mitochondria. HEK-293 cells stably expressing GFP targeted to the ER, co-expressing mitochondrially targeted DsRed, were stimulated with either CCh (i) or Tg (ii). Mobility, expressed as the MI, is shown for the ER (continuous line) and mitochondria (broken line) respectively. Also see Supplementary Movie 2 (http://www.BiochemJ.org/bj/392/bj3920291add.htm).