# A biophysical approach to elucidating the molecular mechanisms of mitotic inhibitor targets

> **NIH NIH K00** · ROCKEFELLER UNIVERSITY · 2021 · $104,674

## Abstract

Many chemotherapy drugs target microtubules in order to inhibit mitosis and stop the proliferation of
cancer cells. Some mitotic inhibitors alter microtubule assembly kinetics in order to stall or prevent microtubule
growth, while others prevent the attachment of microtubules to the relevant mitotic machinery. However, the
fundamental processes of microtubule growth and microtubule attachment to kinetochores remain poorly
understood. The goal of this proposal is to elucidate how the molecular targets of mitotic inhibitors operate under
normal conditions, and how anti-cancer therapeutics alter their mechanisms.
The central approach utilized in this study is to reconstitute the mitotic machinery in vitro using purified
recombinant proteins. This bottoms-up approach has the advantages of allowing for direct and complete control
over experimental conditions, and of enabling single-molecule measurements to be made using powerful
analytical techniques. In this proposal, a new microscopy technique called interferometric scattering microscopy
(iSCAT) is refined and applied in order to answer mechanism questions centering around microtubules. iSCAT
enables direct visualization of unlabeled microtubules at frame rates up to 50,000 frames per second, and can
measure the position of proteins labeled with a 30-nm gold nanoparticle with 2-nm precision. This highly capable
technique opens new doors for studying fast single-molecule kinetics.
In the first aim of this proposal, a custom iSCAT microscope is constructed and used to discover how
microtubule motors use ATP to produce force. In the second aim, iSCAT is used to track the fates of individual
tubulin subunits within the microtubule lattice in order to quantitatively describe how microtubule dynamic
instability is controlled in the absence and presence of anti-mitotic drugs. In the third aim, new advanced
analytical techniques are used to study how microtubules attach to kinetochores during mitosis. Success in these
aims will elucidate in quantitative detail how chemotherapy targets function, and will advance the treatment
options for a broad array of cancers by guiding the development of new anti-mitotic drugs.

## Key facts

- **NIH application ID:** 10225313
- **Project number:** 5K00CA223018-05
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Keith Joseph Mickolajczyk
- **Activity code:** K00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $104,674
- **Award type:** 5
- **Project period:** 2018-08-13 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10225313

## Citation

> US National Institutes of Health, RePORTER application 10225313, A biophysical approach to elucidating the molecular mechanisms of mitotic inhibitor targets (5K00CA223018-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10225313. Licensed CC0.

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