# Imaging system for quantitative in vitro and in vivo analysis of microtubules, actin, and vesicle trafficking

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA BERKELEY · 2020 · $123,283

## Abstract

Abstract - NO CHANGES FROM ORIGINAL
Microtubules (MTs) exhibit dynamic instability in which they exist in growing, pausing, or shrinking states that
interconvert stochastically. This behavior provides the mechanism by which microtubules assemble into a
seemingly infinite variety of structures that provide countless cellular functions including cell motility, mitosis,
and axonal transport. Numerous microtubule-associated proteins (MAPs) and motors bind to the microtubule
lattice or to the growth-favored “plus” end to regulate microtubule assembly dynamics, organization, and
interactions. Important questions that are still unanswered are: how the ensemble behavior of these proteins
collectively regulates microtubule dynamics and how these activities are regulated through cell-cycle stages
and in different cellular subcellular-compartments. A biochemical cell-extract assay recently developed in the
Barnes laboratory unifies, for the first time, two of the most powerful approaches for studies of microtubule
dynamics: biochemical extract studies and genetics. Cell extracts are made from budding yeast mutants and
dynamics of single microtubules are observed. Since mitosis is a highly conserved process, lessons learned
from these studies are likely to apply generally. Unlike many other assays, this assay uses homologous
sources of tubulin and MAPs, avoiding species incompatibility. Moreover, dynamics of single microtubules are
quantitatively analyzed by highly sensitive Total Internal Fluorescence Microscopy. The three aims are: (1) To
determine how specific MAPs and motors affect microtubule assembly dynamics and to reveal emergent
properties that arise from their combined activities. Extracts will be prepared from wild-type yeast and mutants
of different microtubule dynamics regulators, singly or in combinations, and microtubule dynamics in the
extracts will be quantitatively analyzed to parse the contributions of individual proteins to collective microtubule
dynamics regulation. Using cell-cycle-staged extracts from mutants of MT dynamics regulators, proteins
responsible for programmed changes in microtubule dynamics through the cell cycle will be identified. (2) To
determine how activities of these proteins are coordinately regulated through the cell cycle. For four different
cell-cycle stages, phosphorylation sites on MT dynamics regulators will be mapped by mass spectrometry of
the regulators. Functional importance of the identified cell-cycle-specific phosphorylations will be tested by site-
directed mutagenesis of the target proteins. (3) To analyze dynamic properties of kinetochores on microtubules
in yeast extracts and to determine how kinetochores affect microtubule dynamics. The extract system was
successfully adopted for studies of kinetochore association with, and effects on, microtubules. The kinetochore
proteins Mtw1 and Spc105 associate with microtubules in the assay. Moreover, they are directionally
transported toward microtubule plus ...

## Key facts

- **NIH application ID:** 10132550
- **Project number:** 3R01GM047842-25S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** GEORJANA BARNES
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $123,283
- **Award type:** 3
- **Project period:** 1992-09-30 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10132550

## Citation

> US National Institutes of Health, RePORTER application 10132550, Imaging system for quantitative in vitro and in vivo analysis of microtubules, actin, and vesicle trafficking (3R01GM047842-25S1). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10132550. Licensed CC0.

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