Abstract
The cytoskeleton is conventionally viewed as being composed of three filamentous networks; microfilaments, microtubules, and intermediate filaments. This view is challenged by the findings that metabolic enzymes can form filaments with structural functions. We report that phosphofructokinase-1 (PFK1), the first rate-limiting step of glycolysis, assembles into filaments in vitro and in cells. Transmission electron microscopy (TEM) showed that purified liver PFK1 is mainly tetrameric and occasionally formed short filaments in the absence of substrate. Adding the substrate fructose 6-phosphate (F6P) induced the assembly of predominantly long filaments measuring up to 250 nm. PFK1 filaments were less rigid than actin polymers, displaying right angles in contiguous assemblies. The filaments were composed of individual tetramers and had a uniform 11 nm width, resembling an organized addition of subunits forming polymers. Regulated assembly into filaments was also indicated by light scattering measurements that showed a rapid substrate-dependent increase in scattering followed by a stable plateau. Increased light scattering was blocked by excess ATP, which inhibits PFK1 activity. To further confirm activity-dependent filament assembly we generated an inactive but tetrameric liver PFK1 mutant, His199Tyr, and found that in the presence of F6P it does not form filaments, as determined by TEM, or show an increase in light scattering. To assess filament formation by PFK1 in cells, we expressed GFP-tagged PFK1 and used live-cell imaging to examine GFP-PFK1 dynamics. Confocal microscopy reveled that cytosolic PFK1 was recruited to the distal margin of lamellipodia that were devoid of mitochondria. TIRF microscopy reveled that GFP-PFK1 formed dynamic punctae. These data indicate that active but not inactive PFK1 assembles into tetramer-aligned filaments. The activity-dependent recruitment and assembly of PFK1 filaments at the plasma membrane could provide a scaffolding and structural framework for localized ATP production in lamellipodia that lack mitochondria.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.