The influence of blocking actin on PMMA motility function

Although the velocity of the filaments on these samples is much higher than that seen in the previous experiment in chapter 5, the proportional increase in velocity from 0 – 8 kV/m is almost identical. This again highlights the value of the electrical motility device in testing the protein binding mechanism on different surface chemistries. Previous studies have

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presented statistics to show the motility characteristics observed on a single set of samples on multiple surface chemistries.9 However, as can be evidenced by the large amount of papers that have studied myosin on various different surfaces, the velocities on any one surface chemistry can be slightly different from sample to sample.10-13 Imagine for a moment that this study had only taken the characteristics from motility without the effect of a field. In the previous chapter the average velocity of filaments in the absence of a field for PMMA was around 8 µm/s where as TMCS showed an average filament velocity of around 4.5 µm/s at 0 V/m. Taking this statistic alone one might be inclined to think that the PMMA is

outperforming the TMCS in terms of protein binding characteristics that favour retaining full motor protein function. However, if we add an external force into the equation, in this instance an electrical field, we can now analyse how the protein layer reacts to this force by studying the effect on filament motion. The statistics then start to give a very different picture to the binding characteristics of the different surface chemistries, one that would not have been possible to come to without significantly increasing the number of samples, experiments and data collected in the absence of this variable.

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Figure.6.11. The graph shows the velocity of actin filaments when exposed to an electrical field on PMMA. Each line represents assays done with the inclusion of blocking actin (squares) and when the motility assay was run in the absence of blocking actin (triangles). Each data point is an average of 25 filaments movement over 3 samples.

Table 6.6 shows that there is a modest density of ATP inactive HMM bound to the surface. This is shown in the samples that were performed in the absence of blocking actin, as even at 8 kV/m, the percentage of motile filaments is below 80%, which is below that of the other surfaces. The small increase in the percentage of motile filaments, in the case of the assays not treated with blocking actin, would suggest that there is still a significant enough density of inactive motors anchoring the filaments to the surface that even a high field, large force, cannot dislodge them.

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Figure.6.12. The graphs show the angle of trajectory of filaments when an electrical field is applied to an assay made with a PMMA surface. The white bars show the samples that inclusive of blocking actin while the grey bars are samples run without blocking actin. 0 degrees represents the position of the positive electrode. Each graph represents the tracking of 25 filaments over 3 samples.

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Both sets of samples, both blocked and unblocked, have similar directionality characteristics presented in Figure 6.12 showing the angle of trajectory of filament movements. A slight difference is seen, with an increase in the percentage of filament movements towards to the positive electrode evident on the non-blocked samples compared with the blocked samples at the same fields. Couple this relationship with the low increase in velocity seen from 0 – 8 kV/m and the speed at which the filaments returned back to their original velocity in the ‘deceleration’ study, the protein layer on PMMA seems to have a large number of HMM bound to the surface that are unable to propel the filaments. These could be a number of orientations that either trap filaments, as is the case with ATP inactive HMM, or simply block the path of a filament which could result in either diversion or termination of movement.

As the field was increased to 8 kV/m on the samples that had been treated with blocking actin, the percentage of motile filaments is relatively high (not all that far from TMCS). This would indicate that while there is a large portion of ATP inactive HMM bound to the

surface, these are being blocked by the unlabelled actin filaments. Once this has been achieved the obstacle of the extra bound non motile filaments hinder the motility function less than the presence of unblocked ATP inactive HMM. In this case the motile filaments only deal with the crowding caused by the extra filaments on the surface, which at 8 kV/m would seem to have been overcome by the force of the field. Therefore it seems that filaments that are trapped or lodged by overcrowding of the protein layer become dislodged much more easily than those trapped at inactive motor sites.

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Table.6.6. The percentage of actin filaments for the samples with and without blocking actin on PMMA at the tested electrical field strengths. Data represents the tracking of 25 filaments over 3 samples.

Percentage of motile filaments

Field (V/m) 0 4000 6000 8000

PMMA 85.5 88.7 86.8 88.8

PMMA No block 72.9 72.2 78.8 79.6