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Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles
Published on: May 5, 2022
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The glycolytic enzyme phosphofructokinase-1 assembles into filaments
Bradley A Webb1, Anne M Dosey2, Torsten Wittmann1
1Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA.
The Journal of Cell Biology
|June 25, 2017
Summary
Phosphofructokinase-1 (PFK1), a key glycolytic enzyme, forms filaments in a tetramer- and substrate-dependent manner. This discovery reveals new insights into the spatial organization of glucose metabolism within cells.
Area of Science:
- Cellular Biology
- Biochemistry
- Metabolic Pathways
Background:
- Limited understanding of spatial organization for glycolytic enzymes.
- Emerging evidence of non-glycolytic enzymes forming polymers.
Purpose of the Study:
- Investigate filament assembly of phosphofructokinase-1 (PFK1).
- Determine the role of PFK1 isoforms in filament formation.
- Elucidate the mechanism and cellular localization of PFK1 filaments.
Main Methods:
- Recombinant protein expression and purification (PFKL, PFKP, PFKM).
- Negative-stain electron microscopy for filament visualization.
- Biochemical analysis using PFKL/PFKP chimera.
- Live-cell imaging to quantify dynamic properties.
Main Results:
- Liver PFK1 (PFKL) isoform, but not PFKP or PFKM, forms filaments.
- Filaments are apolar, composed of stacked tetramers with exposed catalytic sites.
- The PFKL regulatory domain mediates filament assembly.
- Dynamic PFKL puncta are observed, enriched at the plasma membrane.
Conclusions:
- PFK1 exhibits novel filament assembly behavior.
- Spatial organization of PFK1 impacts cellular glucose metabolism.
- Isoform-specific differences in PFK1 assembly and localization are significant.
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