In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.

Abstract

FCCP (carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone), a potent uncoupler of oxidative phosphorylation, induces the complete disruption of cellular microtubules. A further analysis of this effect on BHK21 cells has shown that a decrease in the number of microtubules can be observed 15 min after adding FCCP and there is complete disruption after 60 min. Regrowth of microtubules was initiated 30 min after removal of FCCP, in marked contrast with the rapid reversion observed when microtubules are disrupted by nocodazole. A similar delay was required for the recovery of mitochondrial function as assessed by rhodamine 123 labelling. The effect of FCCP on microtubules was partially inhibited by preincubation of the cells with NaN3, suggesting that FCCP acts on microtubules through mitochondria. FCCP did not depolymerize microtubules of cells permeabilized with Triton X‐100. In vitro polymerisation of microtubule protein was only slightly diminished by concentrations of FCCP which provoke complete disassembly in vivo. SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis of the microtubules polymerized in vitro in the presence of FCCP showed a reduced amount of high mol. wt. proteins, mainly MAP 2, associated with them. In an attempt to reproduce the mitochondrial effects of FCCP in vitro, we checked the effects of alkaline pH and calcium on microtubule protein polymerization in the presence of FCCP. FCCP did not influence the calcium inhibitory effect but did significantly increase the inhibitory effect of alkaline pH. We conclude that FCCP could depolymerise microtubules in vivo through a dual operation: increasing the intracellular pH by the disruption of the mitochondrial H+ gradient and decreasing the stability of microtubules by impairing the binding of microtubule‐associated proteins.

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“…Once the mitochondria are depolarized, the dye leaks out and disperses throughout the cytosol and yields minimal fluorescence upon excitation. P-triflouromethoxyphenylhydrazone (FCCP) transports protons across mitochondrial inner membranes and induces the depolarization of mitochondria potential (49). Consistent with previous studies (46)(47)(48), our results also suggest that IFI6-16 stabilizes the mitochondrial membrane potential, as shown by residual TMRM fluorescence signal even in cells treated with a low FCCP dose for 10 min (Fig.…”

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

“…A 1 h treatment with FCCP, an uncoupler of oxidative phosphorylation that depolarizes the mitochondrial membrane and abolishes the microtubular network (see below and ref. 27), had no effect on centrosomal GFP-VDAC3, despite diminishing both cytosolic and mitochondrially localized GFP-VDAC3 as well as Mitotracker labeling (Fig. 2B).…”

The pathways of mitophagy for quality control and clearance of mitochondria

“…In addition, CCCP is a nonspecific ionophore that affects cellular components other than the mitochondria. In fact, CCCP causes disruption of the microtubule cytoskeleton 87 and prevents lysosomal acidification. 88 For gentler disruption of mitochondrial function, several other approaches have been used.…”