Quantitative Characterization of Filament Dynamics by Single-Molecule Lifetime Measurements
This chapter discusses some of the current experimental methods used for obtaining quantitative information about the dynamics of filaments by measuring the lifetime of their molecular components. This study focuses specifically on microtubules, filaments found in spindles and composed of tubulin molecules. Microtubules can polymerize and depolymerize tubulin molecules on their ends, and it is this growth and shrinkage process that provides some of the major function of the microtubules. However, there is still much that remains to be understood about how these filmanents grow and shrink. Single-molecule lifetime measurements provide a method to understand the behavior of the filaments by studying the individual tubulin molecules.
The discussion focuses specifically on imaging cytoskeletal molecules- actin and microtubulues, filamanets that can polymerize and depolymerize at their ends. Xenopus extract spindles were imaged by first labeling them with a fluorescent dye that attached to the individual tubulin molecules and then imaging using spinning-disk confocal microscopy. Taking movies of the system reveals that the tubulin molecules are in constant movement. To perform data analysis one needs to carry out particle tracking. To do this, particles must first be identified in each time frame, and then must be linked from frame to frame in order to create a trajectory for each particle.
This particular study was looking at the dissapearance and appearance of tubulin molecules. When they appeared, this indicated that a tubulin molecule was incorporated into the spindle, and when one disappeared this meant that the corresponding molecule had been released from the spindle. By tracking how long a tubulin molecule stays in the microtubule for one can measure the single-molecule lifetime (SLT) and thus get information about microtubule function and formation. One of the reasons measuring SLT is that it can be used to distinguish between different models of microtubule growth.
These models of microtubule growth generally are related to what is called the first-passage time (FPT). In a 1-d system, the FPT would be the time for a microtubule, after growing past a length L, to shrink back to size L (i.e. the first time the microtubule comes back to L). FPT are SLT are related rigorously as shown in the graph below.
These models generally predict an p(x,t), the probability that the end of the microtubule is at coordinate x at time t. The trick for making these SLT measurements relevant is to extract the FPT predicted by a particular model. The chapter discusses five different theories of microtubule growth/shrinkage, and shows for each how to predict what SLT measurements would look like if the model were correct.
Results and Conclusion
SLT measurements were made for the spindles and shown to be consistent with one of the models, 'diffusion-with-drift', and was inconsistent with another one of the models mentioned. Thus this method of single-molecule lifetime measurements can be a useful tool for better understanding the dynamics of filaments such as microtubules.