By measuring the number of myosins in each cluster, the transition rates between them can be determined. (a) Using the transition rates measured for regulated thin filaments (RTFs), a transition probability table was constructed, where the rows are the starting cluster size and the columns, the final. The central diagonal is zero because this table measures the probability of leaving that cluster; all numbers to the right of the diagonal are binding events, and to the left are detachments. Data were obtained at 5 and 10 nM GFP-tagged myosin-S1 (S1-GFP), pCa 6, and 0.1 µM adenosine triphosphate (ATP) and pooled for analysis. A total of 23 kymographs were used to yield 8140 transitions for this analysis. (b) Using the probability of association empirically determined from (a) and known kinetic parameters that govern myosin release, it is possible to construct an expected table of transition probabilities (see ‘Discussion’ for more details). The overall pattern of behaviour between measured (a) and predicted (b) transition probabilities is similar to the right of the central diagonal; however, to the left, there is increased probability of myosin release. (c) The transition probabilities for tropomyosin alone follow a similar pattern to that of the regulated thin filament, indicating that the accelerated release of myosin is mediated mostly by tropomyosin. Data were obtained at 5, 15, and 20 nM S1-GFP in 0.1 µM ATP and pooled for analysis. A total of 19 kymographs were used to yield 2332 transitions for this analysis. Data are available in Figure 4—source data 1.