Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

Abstract

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.

Fig. 1.

Cellular localization of hFGAMS-GFP participating in de novo purine biosynthesis. (A) De novo purine biosynthetic pathway transforms phosphoribosyl pyrophosphate (PRPP) to inosine monophosphate (IMP) in 10 steps. AIRS, aminoimidazole ribonucleotide synthetase; AICAR Tfase, aminoimidazole carboxamide ribonucleotide transformylase; ASL, adenylosuccinate lyase; CAIRS, carboxyaminoimidazole ribonucleotide synthase; FGAMS, formylglycinamidine ribonucleotide synthase; GAR, glycinamide ribonucleotide synthetase; GARS, GAR synthetase; GAR Tfase, GAR transformylase; IMPCH, IMP cyclohydrolase; PPAT, PRPP amidotransferase; and SICARS, succinylaminoimidazole carboxamide ribonucleotide synthetase. Steps 2, 3, and 5 are catalyzed by a trifunctional enzyme, TrifGART; steps 6 and 7 are catalyzed by a bifunctional enzyme, PAICS; and steps 9 and 10 are catalyzed by a bifunctional enzyme, ATIC. (B and C) Distribution of hFGAMS-GFP transiently expressed in HeLa cells grown in purine-rich (B) and purine-depleted (C) media. (Scale bar, 10 μm.)

Fig. 2.

Localization of purinosomes and actin filaments in fixed HeLa cells grown in purine-depleted medium. (A) Transiently expressed and subsequently fixed hFGAMS-GFP–forming clusters in the cytoplasm (green channel). (B) Actin networks stained by rhodamine-phalloidine in the cytoplasm (red channel). (C) Merged image of hFGAMS-GFP (A, green in C) and actin cytoskeleton (B, red in C). (Scale bar, 10 μm.)

Fig. 3.

Subcellular localization of purinosomes harbored by microtubule filaments in HeLa cells grown in purine-depleted medium. (A) Microtubule networks stained by a TubulinTracker Green reagent in the cytoplasm (green channel). (B) Transiently expressed hFGAMS-OFP–forming clusters in the cytoplasm, representing formation of purinosomes (red channel). (C) Merged image of microtubules (A, green in C) and hFGAMS-OFP (B, red in C). (D) Representative region of interest highlighted in the white box in panel (C). Of note, the enlarged image of panel (D) was enhanced for clarification by adjustments of brightness, contrast and/or color balance. (Scale bar, 10 μm.)

Fig. 4.

Effects of small molecules on purinosome assembly formed in HeLa cells grown in purine-depleted medium. Cytochalasin D (A and B) or nocodazole (C and D) was supplied to HeLa cells displaying purinosomes formed by hFGAMS-GFP. Individual images were taken before addition of the inhibitors (untreated; A and C) and after the cells had been incubated with the inhibitors for a given time (B, 90 min; D, 60 min). (Scale bar, 10 μm.)

Fig. 5.

Metabolic flux measurement of de novo purine biosynthesis for HeLa cells. (A) De novo purine biosynthesis is measured by determining amount of [¹⁴C(U)]-glycine incorporated into cellular purines in HeLa cells cultured in purine-rich (■) and purine-depleted (□) media. Incorporation was found to be linear with time up to 4 h, and ratio of de novo purine biosynthesis rates in purine-depleted to purine-rich media was 1.42 by fitting data with the least-squares line method (10, 11). However, data could alternatively be fit with a single exponential function, resulting in larger difference between the two data sets (i.e., 1.60). Error bar indicates SD of three independent assays. Of note, the data points at t = 0 from the two cell culture conditions overlap. (B) Effects of nocodazole on de novo purine biosynthesis was evaluated in a similar way by measuring [¹⁴C(U)]-glycine incorporation for 3 h (Fig. S1). For each type of cells, de novo purine biosynthesis was compared in the absence (i.e., DMSO) and presence of nocodazole. Purine biosynthesis in purine-depleted HeLa cells was decreased by ∼36% in the presence of nocodazole at 3 h. Bar height is the ¹⁴C incorporation into purines per million cells. Error bar indicates SD of three independent assays. *Unpaired one-tailed Student t test revealed that the effect of nocodazole on purine-depleted HeLa cells was statistically significant (P < 0.001). It should be noted that the cells for Fig. 5A were maintained in the preferred growth medium until harvesting, whereas the cells for Fig. 5B were rinsed with buffered saline solution to be treated with nocodazole and then maintained in buffered saline solution until harvesting, to be consistent with cellular imaging conditions.