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Quantification of microtubule (MT) dynamic instability (DI) is essential to mechanistic dissection of MT assembly and the activities of MT binding proteins. Typical methods for quantifying MT dynamics assume that MT behavior consists of growth and shortening phases, with instantaneous transitions (rescues and catastrophes) in between. However, examination of DI data at high temporal and spatial resolution reveals the presence of ambiguous behaviors that cannot easily fit into these categories. Failure to objectively recognize and quantify these behaviors could reduce the reproducibility of DI data and impact attempts to dissect mechanisms. To address these problems, we recently developed STADIA (Statistical Tool for Automated Dynamic Instability Analysis), a MT analysis software package that uses length-history data as input and is (presently) implemented in MATLAB. STADIA uses machine learning methods to objectively analyze and quantify macro-level DI behaviors exhibited by MTs, including variable rates of growth and shortening and a newly quantified DI phase: stutter. Here we overview the process of using STADIA to quantify MT dynamics and provide a set of concrete protocols for using STADIA to process and analyze MT length history data.
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Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe
Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term “stutters.” During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.
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- Award ID(s):
- 1817966
- PAR ID:
- 10336614
- Editor(s):
- Walczak, Claire
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 33
- Issue:
- 3
- ISSN:
- 1059-1524
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1091/mbc.E20-06-0348
