  Protein assembly plays an important role throughout all phyla of life, both
physiologically and pathologically. In particular, aggregation and
polymerization of proteins are key-strategies that regulate cellular function.
In recent years, methods to experimentally study the assembly process on a
single-molecule level have been developed. This progress concomitantly has
triggered the question of how to analyze this type of single-filament data
adequately and what experimental conditions are necessary to allow a meaningful
interpretation of the analysis. Here, we developed two analysis methods for
single-filament data: the visitation analysis and the average-rate analysis. We
benchmarked and compared both approaches with the classic dwell-time-analysis
frequently used to study microscopic association and dissociation rates. In
particular, we tested the limitations of each analysis method along the lines
of the signal-to-noise ratio, the sampling rate, and the labeling efficiency
and bleaching rate of the fluorescent dyes used in single-molecule fluorescence
experiments. Finally, we applied our newly developed methods to study the
monomer assembly of actin at the single-molecule-level in the presence of the
class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our
data indicated fast elongation circumventing a nucleation phase whereas, for
spire, we found that the four WH2 motifs are not sufficient to promote de novo
nucleation of actin.
