# Biomechanics of molecular machines and multiscale non-linear systems

> **NIH NIH R35** · UNIVERSITY OF PENNSYLVANIA · 2024 · $643,500

## Abstract

We work to determine the fundamental principles underlying the operation of molecular machines that give cells the
remarkable ability to segregate their chromosomes during cell division. Various force-sensitive interactions are essential
for mitotic fidelity, and are therefore critical to our understanding of aneuploidy and genomic instability. Over the last 5
years, we have developed molecular tools, equipment, and expertise to quantitatively and rigorously address central
questions about the role of force in chromosome segregation: (1) How do the macromolecular complexes that constitute
human kinetochores travel with dynamic microtubule ends under load? (2) How do individual microtubule-associated
proteins with no motor activity glide along microtubules under dragging force? (3) How does tension applied to the
centromeric chromatin meshwork shape the spatial phosphorylation gradients that orchestrate assembly of the
kinetochores and their binding to microtubules? We approach these problems using reductionist approaches and
innovative in vitro assays that reconstruct these interactions at multiple scales, and analyze our findings with advanced
theoretical modeling. (1) To recreate force-sensitive interactions between microtubules and human kinetochores, we
developed a novel approach for generating macromolecular kinetochore subcomplexes using inducible protein-fusion
scaffolds. When isolated from mitotic HeLa cells, these particles exhibit key physiological properties of native
kinetochores, including their persistent association with dynamic microtubule ends. This breakthrough will enable us for
the first time to study the motility of native human kinetochore complexes, driving forward our biophysical analysis of
kinetochore load-bearing. (2) We will investigate the force sensitivity of individual microtubule-binding proteins at the
single-molecule and ensemble levels using an advanced force spectroscopy approach. We have implemented a highly
sensitive dual-trap, three-bead assay employing an ultrafast force-clamp that allows us to pull on a single non-motor
molecule diffusing on the microtubule wall, imitating the forces these kinetochore-bound molecules experience during
chromosome motions. This approach will provide unique molecular-mechanical insights into the friction-generating
interface that allows the kinetochore to glide along microtubule, while preventing it from slipping from microtubule ends.
(3) We will seek to understand how mechanical deformations shape chemical gradients formed within the chemo-
mechanical meshworks, such as of the centromeric chromatin. Previously, we reconstructed a non-linear Aurora B
kinase/phosphatase bi-stable switch using soluble components. In a proof-of-principle study, we will embed these
enzymatic components into a flexible meshwork to test whether its deformations can control formation of distinct
phosphorylation patterns. Spatio-temporal regulation of the phosphorylation status of kinetochore protei...

## Key facts

- **NIH application ID:** 10833587
- **Project number:** 5R35GM141747-04
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Ekaterina L Grishchuk
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $643,500
- **Award type:** 5
- **Project period:** 2021-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10833587

## Citation

> US National Institutes of Health, RePORTER application 10833587, Biomechanics of molecular machines and multiscale non-linear systems (5R35GM141747-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10833587. Licensed CC0.

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