Dynamic compartmentalization of purine nucleotide metabolic enzymes at leading edge in highly motile renal cell carcinoma

Abstract

Compartmentalization is vital for biological systems at multiple levels, including biochemical reactions in metabolism. Organelle-based compartments such as mitochondria and peroxisomes sequester the responsible enzymes and increase the efficiency of metabolism while simultaneously protecting the cell from dangerous intermediates, such as radical oxygen species. Recent studies show intracellular nucleotides, such as ATP and GTP, are heterogeneously distributed in cells with high concentrations at the lamellipodial and filopodial projections, or leading edge. However, the intracellular distribution of purine nucleotide enzymes remains unclear. Here, we report the enhanced localization of GTP-biosynthetic enzymes, including inosine monophosphate dehydrogenase (IMPDH isotype 1 and 2), GMP synthase (GMPS), guanylate kinase (GUK1) and nucleoside diphosphate kinase-A (NDPK-A) at the leading edge in renal cell carcinoma cells. They show significant co-localization at the membrane subdomain, and their co-localization pattern at the membrane is distinct from that of the cell body. While other purine nucleotide biosynthetic enzymes also show significant localization at the leading edge, their co-localization pattern with IMPDH is divergent. In contrast, a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), predominantly localized in the cytoplasm. Mechanistically, we found that plasma membrane localization of IMPDH isozymes requires active actin polymerization. Our results demonstrate the formation of a discrete metabolic compartment for localized purine biosynthesis at the leading edge, which may promote localized nucleotide metabolism for cell migration and metastasis in cancers.

Keywords: GMPS; GUK1; IMPDH; Metabolic compartmentalization; Salvage purine synthesis; de novo purine synthesis.

Figure 1:. IMPDH1 and IMPDH2 localize to the leading edge of RCC cells.

Immunofluorescence staining of IMPDH1 (left) and IMPDH2 (right) in Caki-1 (A) and 786-o (D) cells. Insets of leading edge in pseudocolor shown below. Intensity measurements of immunofluorescence staining at leading edge of Caki-1 (B) and 786-o (E) cells. Ratio of fluorescence intensity at leading edge membrane compared to total cell membrane for Caki-1 (C) and 786-o (F) cells. Scale bars indicate 10 μm.

Figure 2:. Nucleotide metabolic enzymes localize at the lamellipodial membrane.

Purine nucleotide metabolism pathway. De novo enzymes (orange), ATP biosynthetic branch (green), and GTP biosynthetic branch (blue) (A). Fluorescence imaging for GTP biosynthetic enzymes GMPS (mCherry-tagged, 786-o), GUK1 (Myc, Caki-1), and NDPK-A (antibody, Caki-1) (B). Immunofluorescence staining for de novo enzymes PRPS1 (Myc, Caki-1), FGAMS (GFP, Caki-1), PAICS (GFP, Caki-1), ADSL (GFP, 786-o), and ATIC (GFP, Caki-1) (C). Insets of leading edge in pseudocolor shown below. Scale bars indicate 10 μm. Asterisk (*) indicates enzymes involved in multiple pathways. PRPS: phosphoribosyl pyrophosphate synthetase. PPAT: phosphoribosyl pyrophosphate amidotransferase. GART: phosphoribosylglycinamide formyltransferase. FGAMS: phosphoribosyl formylglycinamidine synthase. PAICS: phosphoribosyl aminoimidazole succinocarboxamide synthetase. ADSL: adenylosuccinate lyase. ATIC: 5-amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase. ADSS: adenylosuccinate synthase. AK: adenylate kinase. IMPDH: inosine-5′-monophosphate dehydrogenase. GMPS: GMP synthase. GUK1: guanylate kinase 1. NDPK: nucleoside-diphosphate kinase. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Figure 3:. Purine nucleotide enzymes compartmentalize at the leading edge.

IMPDH1 (green) (A) and IMPDH2 (red) (B) co-localize with selected enzymes at the leading edge but not within the lamellum. Correlation scores of co-stained enzymes are an average 2.7-fold higher at the leading edge compared to the cell body (C). Insets shown at right. Scale bars indicate 10 μm.

Figure 4:. Actin polymerization is required for IMPDH localization to the membrane.

IMPDH1 (green) (A) and IMPDH2 (green) (B) co-localize with WGA (red) in DMSO-treated conditions (top row); 10 μM Lat B treatment for 2 h (bottom row) decreases co-localization. Confocal microscopy. Insets shown at right. Scale bars indicate 10 μm. WGA: wheat germ agglutinin.