Actin-ATP hydrolysis is a major energy drain for neurons

Abstract

In cultured chick ciliary neurons, when ATP synthesis is inhibited, ATP depletion is reduced approximately 50% by slowing actin filament turnover with jasplakinolide or latrunculin A. Jasplakinolide inhibits actin disassembly, and latrunculin A prevents actin assembly by sequestering actin monomers. Cytochalasin D, which allows assembly-disassembly, but only at pointed ends, is less effective in conserving ATP. Ouabain, an Na(+)-K(+)-ATPase inhibitor, and jasplakinolide both prevent approximately 50% of the ATP loss. When applied together, they completely prevent ATP loss over a period of 20 min, suggesting that filament stabilization reduces ATP consumption by decreasing actin-ATP hydrolysis directly rather than indirectly by modulating the activity of Na(+)-K(+)-ATPase, a major energy consumer.

Fig. 1.

a, Slowing filament turnover conserves ATP. Time-lapse imaging of ciliary neurons, loaded with MgGr, was used to monitor ATP depletion after a block in ATP synthesis. As ATP is depleted, [Mg²⁺]_i increases. Analysis includes all cells in 20 fields. Similar results were seen in two replicas of this experiment; mean ± SEM of >100 cells.b, c, Phase micrographs of fixed neurons before (b) and 40 min after (c) application of ATP-synthesis block (10 mm NaN₃ to 6 mm 2-deoxyglucose). Chemically ischemic cells show extensive neurite retraction, somata shrinking, and rounding, lysed cell debris (arrowheads), and vacuoles (arrows). Scale bar, 30 μm.jas, Jasplakinolide; lat A, latrunculin A; cyto D, cytochalasin D.

Fig. 2.

The minimum jasplakinolide concentration needed to stabilize filaments is >6 nm. a–c, Incubation in as little as 20 nm jasplakinolide (jas) induces a filopodial expansion, indicating stimulation of actin assembly rather than mere stabilization of F-actin, here stained with rhodamine phalloidin. Scale bar, 10 μm.d, Transmitter vesicle recycling depends on filament turnover rate (Bernstein et al., 1998) and is attenuated by preincubation in jasplakinolide >6 nm. Cells were depolarized in a 75 mm K⁺ buffer containing the fluorescent styryl dye FM1-43 (10 μm), which is used to monitor depolarization-induced vesicle cycling (Cochilla et al., 1999). g, Overlay of DIC (e) and fluorescence (f) images, showing depolarization-induced FM1-43 uptake in ciliary calyx. Scale bar, 20 μm.

Fig. 3.

Morphological evidence for promotion of actin disassembly by latrunculin (Lat A) and stabilization of F-actin by jasplakinolide (Jas). a, DIC micrographs of live cells without jasplakinolide pretreatment show lysis and loss of cell shape (flattening) after 15 min of incubation in 1 μm latrunculin A. The dose-dependent nature of jasplakinolide stabilization of cell morphology seen inb is plotted in c. A concentration as low as 8 nm has a stabilizing effect. All cells in >20 fields were included in data plotted for each jasplakinolide concentration.

Fig. 4.

Effects of actin modulators on ATP, Na⁺, and Ca²⁺ levels in ischemically stressed cells. a, Reducing filament turnover moderates ischemically induced increase in [Ca²⁺]_i but is not likely to contribute significantly to ATP conservation of jasplakinolide (jas)- and latrunculin A (lat A)-treated cells. b, Similar time courses were observed for ATP depletion in rat embryonic day 18 brain cortical cells measured with either a cell lysate–luciferase assay or an [Mg²⁺]_i fluorescent dye indicator and for ciliary cells using MgGr or Mag-fura-2; exponential curve fit shown. c, d, Actin assembly modulators conserve ATP via a reduction in actin-ATP hydrolysis rather than a reduction in Na⁺–K⁺-ATPase activity.c, Preincubation in 10 nm jasplakinolide or 1 μm latrunculin A accelerates [Na⁺]_i increase during ATP depletion. Only in ATP-depleted control cells is [ATP] low enough to reverse Na⁺–Ca²⁺ exchanger, pulling Ca²⁺ in and extruding Na⁺. Not unexpectedly, PBS–1.5 mm ouabain, an Na⁺–K⁺-ATPase inhibitor, causes the fastest rise in Na⁺. d, Conservation of ATP by ouabain and jasplakinolide is additive.a–d, Mean ± SEM of two or three experiments;n = 200–300 cells in 60 fields.