# Integrative analyses of the kinetochore and the spindle assembly checkpoint

> **NIH NIH R35** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $530,201

## Abstract

The primary goal of mitosis is to make two genetically identical copies of the dividing cell. To achieve this goal,
the dividing cell must segregate exactly one copy of each chromosome into each daughter. Even a single error
in chromosome segregation results in aneuploidy, which in turn leads to a plethora of defects, from cell death to
tumorigenesis. Therefore, to accomplish accurate chromosome segregation, the eukaryotic cell uses two highly
sophisticated systems: the kinetochore and the Spindle Assembly Checkpoint (SAC). The kinetochore is a multi-
protein machine that moves and segregates each chromosome. If it is unable to do so, the kinetochore activates
the SAC. The SAC is a signaling cascade that generates a diffusible checkpoint complex that arrests cell division.
Extensive research has compiled a nearly complete list of proteins and activities necessary for the two systems.
However, fundamental questions regarding each remain unanswered. How does the kinetochore seamlessly
integrate the disparate molecular mechanisms that generate chromosome movement and activate the SAC?
How does the cell calibrate SAC signaling output to maximize accurate chromosome segregation, but minimize
the duration of mitosis? The most significant challenge in defining the molecular mechanisms of kinetochore
function is its highly complex protein architecture. My lab reconstructed the nanoscale protein architecture of the
kinetochore in budding yeast by developing an array of fluorescence microscopy techniques. We used this
knowledge to undertake `architecture-function' analyses of the yeast kinetochore. Our work reveals how
kinetochore architecture shapes functional mechanisms. Our next goal is to define how the architecture of the
much more complex, human kinetochore shapes emergent mechanisms of force generation and SAC activation.
The most significant challenge in studying the biochemical design of the SAC is our inability to measure the
thermodynamic rate constants governing its signaling reactions. This is because these complex reactions are
localized within the nanoscopic kinetochore. To circumvent this challenge, we designed the “eSAC”: an ectopic,
quantifiable, and controllable, SAC activator. Preliminary characterization of the biochemical design of the SAC
provides an elegant model to explain how the human cell optimizes the SAC signaling cascade. We will use the
eSAC to quantify biochemical steps in the SAC cascade, reconstitute key steps to study them at the
thermodynamic and structural level, and then synthesize a detailed mathematical model to completely establish
the mechanistic platform describing the SAC. Our integrative analyses of the two systems will thus elucidate
their respective functional designs, and reveal how they cooperate to ensure accurate chromosome segregation.

## Key facts

- **NIH application ID:** 10439662
- **Project number:** 5R35GM126983-05
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Ajit Joglekar
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $530,201
- **Award type:** 5
- **Project period:** 2018-07-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10439662

## Citation

> US National Institutes of Health, RePORTER application 10439662, Integrative analyses of the kinetochore and the spindle assembly checkpoint (5R35GM126983-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10439662. Licensed CC0.

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