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Updated: Aug 14, 2026
Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays
Published on: February 4, 2021
Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates
Abstract:
We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.
Insights
Microfilament movement in Physarum polycephalum flagellates depends on intact microtubules. Calcium disrupts microtubules, inhibiting ATP-induced microfilament sliding, suggesting a crucial role for microtubule integrity in this motility.
Area of Science:
- Cell Biology
- Cytoskeletal Dynamics
- Biochemistry
Background:
- Previous observations indicated microfilament displacement over microtubules in Physarum polycephalum.
- The precise mechanism and dependencies of this cytoskeletal interaction remained unclear.
Purpose of the Study:
- To investigate the role of the microtubular cytoskeleton in ATP-induced microfilament movement in Physarum polycephalum flagellates.
- To elucidate the molecular mechanisms underlying the observed motility.
Main Methods:
- Permeabilized Physarum polycephalum flagellates were treated with calcium ions (Ca2+) to disrupt microtubules.
- Subsequent treatment with magnesium-adenosine triphosphate (Mg-ATP) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) assessed microfilament movement.
- Taxol was used to stabilize microtubules, and electron microscopy (EM) examined cytoskeletal structure.
- Pharmacological agents like GTP, ATPγS, ADP, AMP-PNP, vanadate, and EHNA were used to characterize the motility.
Main Results:
- Disruption of microtubules by Ca2+ inhibited ATP- and EGTA-induced microfilament movement.
- Taxol pretreatment protected microtubules from Ca2+ and preserved subsequent microfilament motility.
- EM revealed ATP treatment caused reciprocal sliding between microfilaments and microtubules, not within them.
- Motility was stimulated by ATP, partially by GTP and ATPγS, but inhibited by ADP, AMP-PNP, vanadate, and EHNA.
Conclusions:
- The integrity of the microtubular cytoskeleton is essential for ATP-induced microfilament movement in Physarum polycephalum.
- The observed motility involves reciprocal sliding between microfilaments and microtubules, distinct from other known microtubule-based motors.
- The pharmacological profile suggests a unique motor mechanism, partly resembling but not identical to known microtubule-dependent motilities.
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