Eukaryotic flagella and cilia are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella and cilia contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins, located all along the length of the axonemal structure.
Fluorescent ATP analogs have been a useful tool to study ATPase activity of various motor proteins. \acrfull{mant} has been previously used to probe the activity of various ATPases, including dynein. It has been shown by various authors, that MANT-ATP supports dynein activity as well as the axonemal beat. However, direct observations of binding to the axonemal structure were not previously reported.
Using highly sensitive fluorescent microscopy to monitor the binding of the fluorescent ATP analog, I probed dynein activity directly in the immobilized intact axoneme for the first time.
To understand these kinetics a kinetic model was developed. By fitting this model to experimental data I was able to identify ATP-binding sites with distinct kinetic properties in the axoneme.
I report a turnover rate of k = 0.02 s−1 at 1μM mant-ATP for dynein. Moreover, I discovered that there is binding of the ATP analog to the axoneme with a much higher rate of k = 11 s−1 at 1μM mant-ATP. By the application of this method to axonemes with reduced dyneins, it has been identified that the slow rate belongs to dynein.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:34182 |
| Date | 11 June 2019 |
| Creators | Feofilova, Maria |
| Contributors | Howard, Jonathon, Jülicher, Frank, Sbalzarini, Ivo, Howard, Jonathon, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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