# Saccharomyces cerevisiae microtubule and kinetochore dynamics

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA BERKELEY · 2023 · $401,250

## Abstract

Project Summary/Abstract
For replicated chromosomes to be segregated to two daughter cells accurately, the microtubule (MT)
cytoskeleton must be completely remodeled to form a bipolar spindle. A large, dynamic protein complex called
the kinetochore attaches replicated chromosomes to microtubules emanating from opposite spindle poles.
Proper kinetochore-microtubule attachment is vital for preservation of genomic integrity and prevention of
cancer and birth defects. Therefore, mitotic spindle assembly and kinetochore attachment must be well
coordinated. Challenging understanding of these processes and their coordination is the fact that mitotic
spindles and kinetochores are extremely complex molecular machines (kinetochores contain >60 proteins),
that they are targets of phosphoregulation by multiple protein kinases, and that they possess a striking range of
biochemical activities. Kinetochore activities include: (1) lateral MT binding, (2) translocation along the MT
lattice to the plus end, (3) conversion from lateral to end-binding, (4) association with dynamic MT ends while
tubulin subunits are exchanged, and (5) serving as force-coupling devices between chromosomes and MT plus
ends during anaphase A. Understanding how the kinetochore performs its various functions, the structural
underpinnings of these activities, and how these activities are regulated post-translationally, is far from
complete. A biochemical cell-lysate assay recently developed in the Barnes laboratory combines, for the first
time, two of the most powerful approaches for studies of microtubule dynamics: biochemical extract studies
and genetics. Dynamics of single microtubules and single kinetochores associated with these microtubules are
revealed and quantitatively analyzed by highly sensitive Total Internal Reflection Fluorescence microscopy.
Cell lysates synchronized to specific cell-cycle stages are made from budding yeast mutants of specific mitotic
proteins. Quantitative analysis of MT dynamics and kinetochore activities will establish how these parameters
are regulated in the cell cycle, and these studies will identify the specific proteins that carry out the specific
behaviors. Since mitosis is a highly conserved process, lessons learned from these studies are expected to
apply broadly. Unlike many other assays, this assay exclusively uses homologous sources of tubulin and
interacting proteins, avoiding artefacts that arise from species mismatch incompatibilities. Proposed studies
build upon recent unique observations of microtubule dynamics regulation and kinetochore dynamic activity in
this lysate system. The objectives are: (1) To investigate biochemical activities of intact kinetochores and their
regulation, and to relate kinetochore structure to function; and (2) To investigate how microtubule dynamics are
regulated through the cell cycle in both the nucleus and the cytoplasm, focusing on Kar3 and Kip3 kinesins.

## Key facts

- **NIH application ID:** 10623066
- **Project number:** 1R35GM149237-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** GEORJANA BARNES
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $401,250
- **Award type:** 1
- **Project period:** 2023-06-01 → 2028-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10623066

## Citation

> US National Institutes of Health, RePORTER application 10623066, Saccharomyces cerevisiae microtubule and kinetochore dynamics (1R35GM149237-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10623066. Licensed CC0.

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