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Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations
Published on: September 20, 2019
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A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle
Ailine Stolz1, Norman Ertych, Holger Bastians
1a Georg-August University Göttingen; Göttingen Center for Molecular Biosciences (GZMB) and University Medical Center Göttingen (UMG) ; Institute of Molecular Oncology; Section for Cellular Oncology ; Göttingen , Germany.
Cell Cycle (Georgetown, Tex.)
|January 16, 2015
Summary
Increased microtubule polymerization in cancer cells drives chromosome missegregation. A new assay identifies regulators of microtubule plus end assembly, aiding the discovery of genetic alterations linked to chromosomal instability (CIN).
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Proper regulation of microtubule dynamics is crucial for accurate chromosome segregation during mitosis.
- Increased microtubule plus end assembly rates are observed in human cancer cells, contributing to chromosomal instability (CIN).
- Identifying genetic alterations causing these increased polymerization rates is challenging due to technical limitations in large-scale live-cell screening.
Purpose of the Study:
- To develop a robust phenotypic screening assay for systematically identifying regulators of microtubule plus end polymerization.
- To uncover genetic alterations responsible for increased microtubule polymerization rates in cancer cells.
- To identify novel regulators of microtubule dynamics and their role in chromosomal instability.
Main Methods:
- Development of a novel phenotypic screening assay based on analyzing monopolar mitotic spindle structures induced by Eg5/KIF11 inhibition.
- Assessment of the asymmetric monoaster phenotype caused by increased microtubule polymerization in both live and fixed cells.
- Execution of a small interfering RNA (siRNA) screen to identify regulators of microtubule plus end assembly.
Main Results:
- The developed assay reliably detects increased microtubule polymerization, manifesting as asymmetric monoasters upon Eg5/KIF11 inhibition.
- The siRNA screen identified several proteins, including microtubule plus end binding proteins, centrosomal, and cortex-associated proteins, as regulators of microtubule plus end assembly.
- A subset of identified regulators was found to dampen microtubule plus end polymerization, influencing spindle orientation.
Conclusions:
- The novel phenotypic assay provides a scalable method for identifying regulators of microtubule plus end polymerization.
- The study identified novel regulators of microtubule dynamics, offering insights into mechanisms driving chromosomal instability in cancer.
- Understanding these regulators' roles in microtubule polymerization and spindle orientation is crucial for developing targeted cancer therapies.
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