Author ORCID Identifier

https://orcid.org/0000-0002-5254-4662

Semester

Fall

Date of Graduation

2025

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Bradley Webb

Committee Member

Jianhai Du

Committee Member

Werner Geldenhuys

Committee Member

Michael Robichaux

Committee Member

Michael Schaller

Abstract

Glycolytic enzymes are spatially organized in the cytoplasm, and are shown to localize with cellular structures such as the plasma membrane, F-actin, mitochondria, or associate independently within the cytosol. The assembly of these enzymes is hypothesized to regulate pathway flux to meet the energetic needs of the cell. However, it is unclear whether all of these assemblies have the same mechanism of formation or function in the cell. We investigated the role of glycolytic enzyme assemblies in cells, using the glycolytic “gatekeeper” enzyme phosphofructokinase-1 (PFK1). PFK1 is extensively regulated by multiple mechanisms, including over 20 allosteric ligands, post translational modifications, and assembly state. The liver isoform (PFKL) forms filaments in vitro, and while early characterization suggests they are able to assemble PFKL into punctate assemblies in the cytoplasm, their function in cells remains unknown. In migratory cells, PFKL localizes to lamellipodia in a filament-dependent manner, and disrupting this localization alters directional sensing but not migration velocity. Disrupting filament formation does not alter enzyme activity or glycolytic flux as estimated by extracellular acidification rate (ECAR) in cells, suggesting that the filament-dependent organization of PFKL in cells may have a function beyond local ATP generation in lamellipodia. These results identified a novel function of PFKL filament formation in regulating spatial organization of the enzyme in the cytoplasm. To further investigate the role of PFKL assembly state in cells, a PFK1-null cell line was generated. Loss of PFK1 activity and glycolytic flux from PFKL knockout can be restored by re-expression of exogenous protein, validating the utility of this cell model. Expressing filament-incompetent PFKL-N702T does not alter PFK1 activity or ECAR, suggesting that at least in basal conditions filaments don’t function to alter enzyme activity. Additionally, this cell model was used to provide evidence of a catalytically active PFKL dimer in cells, challenging the requirement of tetramer formation for mammalian catalytic activity. Future experiments will use this cell model to investigate the function of PFKL-containing glycolytic assemblies in the cytoplasm, testing the hypothesis that they form in a filament-dependent manner in response to energetic stress to increase glycolytic flux and improve cell survival. Collectively, this data expands our understanding of the mechanisms underlying the organization of glycolytic enzymes in the cytoplasm by identifying novel mechanisms regulating activity and spatial organization of PFKL via filament formation. This work contributes to the larger body of literature studying the mechanisms and function of spatially organized metabolic enzymes in the cytoplasm.

Download

Share

COinS