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Cellular Assays for Measuring Dynamin Activity in Muscle Cells
Jessica Laiman1, Ya-Wen Liu2,3
1Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
Abstract:
Dynamin is one of the best-studied membrane fission machineries, which mediates endocytic vesicle pinch-off from the plasma membrane. Among the three dynamin isoforms encoded in mammalian genome, dynamin-2 is the ubiquitously expressed isoform and leads to human muscular or neuronal diseases when mutants causing hyperactivity or hypoactivity of its membrane fission activity occur. While transferrin uptake is the most commonly used assay to measure dynamin activity in cultured cells, here we provide two different methods to quantitatively examine the activity of dynamin in myoblasts and myotubes, i.e., Bin1-tubule vesiculation and glucose transporter 4 fractionation assays, respectively. These methods could provide a quantitative measurement of dynamin activity in both differentiated and undifferentiated myoblasts.
Insights
Researchers developed new assays to measure dynamin-2 activity in muscle cells. These methods, focusing on Bin1-tubule vesiculation and glucose transporter 4 fractionation, offer quantitative insights into dynamin function in myoblasts and myotubes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Dynamin is a key protein complex essential for membrane fission during endocytosis.
- Dynamin-2 is the primary isoform in mammals, crucial for cellular processes and linked to muscular and neuronal diseases when dysfunctional.
- Current assays, like transferrin uptake, have limitations in specific cell types.
Purpose of the Study:
- To establish novel, quantitative assays for measuring dynamin activity in muscle cells.
- To assess dynamin-2 function in both undifferentiated myoblasts and differentiated myotubes.
- To provide alternative methods to the standard transferrin uptake assay.
Main Methods:
- Developed the Bin1-tubule vesiculation assay to quantify dynamin-mediated membrane fission.
- Implemented the glucose transporter 4 (GLUT4) fractionation assay to assess dynamin activity.
- Utilized myoblasts and myotubes as model systems for assay validation.
Main Results:
- Successfully demonstrated the utility of Bin1-tubule vesiculation for measuring dynamin activity.
- Validated the GLUT4 fractionation assay as a quantitative method for dynamin assessment in muscle cells.
- Showcased the applicability of both assays in both differentiated and undifferentiated myoblasts.
Conclusions:
- The Bin1-tubule vesiculation and GLUT4 fractionation assays provide robust, quantitative measures of dynamin activity in muscle cells.
- These novel methods overcome limitations of existing assays, offering broader applicability in myoblast research.
- The developed assays facilitate a deeper understanding of dynamin's role in muscle cell physiology and disease.
