Imaging and physically probing kinetochores in live dividing cells

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• The kinetochore is a complex protein assembly essential for accurate chromosome segregation during cell division.
• Understanding kinetochore mechanics and dynamics is vital for comprehending cell cycle regulation and preventing aneuploidy.
• Mammalian kinetochores comprise over 100 protein species, presenting significant challenges for in-situ study.

Purpose of the Study:

• To present novel experimental approaches for imaging and physically probing kinetochores.
• To overcome challenges posed by mitotic cell rounding and rapid kinetochore dynamics.
• To provide detailed methodologies for broader accessibility and application in cell biology research.

Main Methods:

• Mild spindle compression to enhance kinetochore imaging quality.
• Stronger spindle compression to mechanically perturb the spindle and kinetochore complex.
• Simultaneous imaging of dual-color kinetochore reporter probes at subpixel resolution to track structural dynamics.

Main Results:

• Mild spindle compression improves the resolution and clarity of kinetochore visualization.
• Mechanical perturbation via stronger compression allows for the study of kinetochore responses to force.
• Subpixel resolution imaging of reporter probes reveals kinetochore structural dynamics under applied cellular forces.

Conclusions:

• The developed methods offer robust ways to image and mechanically probe kinetochores.
• These techniques facilitate the study of kinetochore function and dynamics in response to cellular forces.
• The approaches are adaptable for investigating other dynamic cellular structures.