Visualizing and Weighing Kinesin-8 Microtubule Depolymerization with Mass Photometry

• Applicant: Dr. Anita Jannasch
• Subject Area: Biophysics
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 537583262

Microtubules are an essential cytoskeletal component and key for cell division, and motility. While intrinsically they are highly dynamic structures, their length is also controlled by other regulators such as motor proteins of the kinesin 8 and 13 family. Since microtubule length regulation is important for mitosis, these motors have been associated with diseases such as cancer. Depolymerizing kinesins do not transport vesicles, but target microtubule ends for their disassembly. While their translocation to the microtubule end is reasonably well understood, how their hydrolysis cycle is coupled to tubulin removal and how many tubulin dimers are removed per end encounter is unclear. Here, we will develop a novel interference reflection microscopy (IRM) setup for (i) label-free tracking of single budding yeast kinesin-8 Kip3 and (ii) simultaneously measuring its mass removal at microtubule ends during depolymerization. Kip3 will be used as a model system for microtubule depolymerases in reconstituted, in vitro assays with purified components. Our novel IRM setup will enable the imaging of single proteins through interference of light scattered from the protein and the sample glass surface. A rolling differential averaging procedure allows us to determine its ratiometric contrast. Since this contrast is directly proportional to the protein’s molecular weight, the IRM microscope is also a mass photometer. Furthermore, the measurement of the center-of-mass allows single-molecule localization with nanometer precision. Preliminary measurements on an IRM mass photometer prototype confirmed the ability to resolve the mass of single tubulin and Kip3 monomers. On a commercial mass photometer microtubule depolymerization by Kip3 could in principle be measured, but the measurement time was limited by photodamage. Other challenges include the significant molecular weight difference between kinesins, tubulins, and microtubules and the surface-distance dependence of the ratiometric contrast. To overcome these challenges, we will develop and optimize a new IRM mass photometry instrument combined with total internal reflection fluorescence (TIRF) microscopy. The optimization will be complemented with computational approaches to improve contrast, particle detection, and account for its surface distance. With the optimized setup, we will be able to track unlabeled motor proteins on microtubules and measure mass removal or addition down to a molecular weight of 10 kDa with about 100-1000 times less light compared to the commercial system. The latter aspect will allow sufficiently long observation times of microtubule depolymerization without photodamage. By resolving the interaction of single Kip3 motors with microtubules and measuring the number of removed tubulins, we will gain insights into the depolymerization mechanism. In the long term, we hope that our work on mictotubule depolymerases will be beneficial for cancer research.

• DFG Programme: Research Grants
• Major Instrumentation: Custom TIRF-IRM
• Instrumentation Group: 5040 Spezielle Mikroskope (außer 500-503)