Live-cell imaging and analysis of actin-mediated mitochondrial fission
Daisuke Shimura1, Robin M Shaw2
1Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Surgery, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA.
Abstract:
Current approaches, such as fixed-cell imaging or single-snapshot imaging, are insufficient to capture cytoskeleton-mediated mitochondrial fission. Here, we present a protocol to capture actin-mediated mitochondrial fission using high-resolution time-lapse imaging. We describe steps starting from cell preparation and mitochondria labeling through to live-cell imaging and final analysis. This approach is also applicable for analysis of multiple cytoskeleton-mediated organelle events such as vesicle trafficking, membrane fusion, and endocytic events in live cells. For complete details on the use and execution of this protocol, please refer to Shimura et al. (2021).1.
Insights
This study introduces a high-resolution time-lapse imaging protocol to capture actin-mediated mitochondrial fission, overcoming limitations of current methods. This technique also enables the study of other cytoskeleton-mediated organelle dynamics in live cells.
Area of Science:
- Cell Biology
- Microscopy
- Biophysics
Background:
- Current imaging techniques fail to adequately capture cytoskeleton-mediated mitochondrial fission.
- Understanding organelle dynamics is crucial for cell function.
Purpose of the Study:
- To present a novel protocol for high-resolution time-lapse imaging of actin-mediated mitochondrial fission.
- To provide a method applicable to various cytoskeleton-mediated organelle events.
Main Methods:
- Development of a detailed protocol for live-cell imaging.
- Mitochondria labeling and cell preparation techniques.
- High-resolution time-lapse microscopy and data analysis.
Main Results:
- Successfully captured actin-mediated mitochondrial fission in live cells.
- Demonstrated the protocol's versatility for studying other organelle dynamics like vesicle trafficking and endocytosis.
Conclusions:
- The presented time-lapse imaging protocol offers a significant advancement for studying cytoskeleton-mediated mitochondrial fission.
- This methodology is broadly applicable to live-cell organelle dynamics research.
