Maximum limit to number of Myosin II motors participating in an ensemble motility

Molecular motor myosin II participates in muscle contraction, actin cortex remodeling and cytokinesis ring constriction. Myosin II is an ensemble motor i.e., millions of Myosin II work in parallel in muscle to produce simple movements like blinking of an eye. It has been an interesting puzzle for scientific community to understand the allosteric rules between molecules that regulate/co-ordinate motors when they work in group. 
To understand ensemble behavior of Myosin II motor, we purified motors from chicken breast muscle and reconstituted in an in vitro motility assay. In this assay fluorescently labelled actin filaments slide over carpet of myosin molecules without any load and can be compared with unloaded shortening of muscle contraction. We performed this assay at different myosin density and ATP concentrations. By changing immobilized myosin density and ATP concentration we change the number myosin molecules available for interaction/unit length of actin filament. Actin filaments of >10 micron length were added in flow cell and assay was initiated with addition of ATP. All the actin filaments break down in smaller pieces within minutes after addition of ATP.  Average length of sliding filaments correlates well with motors density in the surface and also to ATP concentrations in solution. Filament length increases with decrease in density of motors and decreases at low ATP concentration.
Actin filament breaks due to bucking between force generating heads and resisting heads. We developed a probility model to calculate number of interacting heads using kinetic parameters of Myosin ATPase cycle. Number of resisting heads are mainly due to rigor heads that are waiting for binding of ATP and detach. These rigor heads in an ensemble increase at higher myosin density or lower ATP concentration. Based on this data, we suggest there is an upper limit to number of Myosin II participating in ensemble motility. 

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Compact TIRF Demo
Evanescent wave generated by total internal reflection of light is useful method for exciting fluorescent molecules close to surface. Traditionally TIRF excitation has been done using high numerical aperture objective and coherent Laser lights. We have developed a compact TIRF module that can be used as attachment to any existing microscopes and image with high S/N ratio. A simple demonstration of the compact TIRF module will be shown.