Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts

21-POS

Board 21

Modifying Actomyosin Function by Small-molecular Inhibitor Blebbistatin

Mohammad A. Rahman

, Alf Månsson.

Linnaeus University, Kalmar, Småland, Sweden.

Addition of small molecular substances could substitute genetic engineering for temporarily

changing myosin motor function. One interesting compound is blebbistatin that inhibits myosin

II motor activity. In muscle cells, the effect of actin-myosin sliding velocity is modulated by

myosin regulatory light chain (RLC) phosphorylation1. Otherwise, evidence exists that

blebbistatin stabilizes a start of the power-stroke, strongly attached, actomyosin state2. Here, in

vitro motility assay studies are performed to investigate (a) whether the drug effect varies with

myosin RLC phosphorylation in absence of the myofilament lattice of muscle and (b) if the

effect on velocity is consistent with stabilization of the mentioned actomyosin state. Heavy

meromyosin (HMM) from Rabbit skeletal muscle, with either phosphorylated or un-

phosphorylated RLCs, was adsorbed to silanized surfaces for in vitro motility assays. We found

that blebbistatin (1µM) inhibits sliding velocity to similar degree (~50 %) independent of

phosphorylation status. The inhibition induced by blebbistatin (2µM) was doubled for an

increase in ionic strength (60 to 130 mM) whereas the effect on velocity was similar at different

MgATP concentrations (0.1 – 1 mM). The findings suggest that the blebbistatin effect on

velocity does not depend on myosin RLC phosphorylation, consistent with the idea that such

dependence in vivo is fully attributed to myosin head – backbone interactions (absent in vitro).

Furthermore, modelling suggests that blebbistatin increases the population of a start-of-power

stroke actomyosin state with possible physiological roles e.g. in eccentric contractions (stretch of

active muscle). 1.Stewart, M., Franks-Skiba, K. & Cooke, R. (2009) J. Muscle Res. Cell Motil.

30, 17–27.2.Takács, B. et al. (2010). Proc. Natl. Acad. Sci. 107, 6799–6804 Supported by the

FET-program of EU-FP7 (grant agreement 613044; ABACUS)