Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

Songon Ana, Yijun Denga, John W. Tomshoa, Minjoung Kyoungb, and Stephen J. Benkovica,1

aDepartment of Chemistry, Pennsylvania State University, University Park, PA 16802; and bDepartment of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305

Contributed by Stephen J. Benkovic, June 15, 2010 (sent for review April 29, 2010)

Evidence has been presented for a metabolic multienzyme complex, Results the purinosome, that participates in de novo to Microtubule-Associated Purinosome Formation. We investigated form clusters in the cytoplasm of living cells under purine-depleted whether cytoskeletal structures are involved in the purinosome as- conditions. Here we identified, using fluorescent live cell imaging, sembly. First, we stained actin filaments with rhodamine-phalloidine that a microtubule network appears to physically control the spatial in fixedHeLacellsandalsomicrotubuleswithaTubulinTracker distribution of purinosomes in the cytoplasm. Application of a cell- Green reagent in live HeLa cells in the presence of hFGAMS-GFP/ based assay measuring the rate of de novo purine biosynthesis OFP as a purinosome marker. The cytosolic clusters did not coloc- fi fi con rmed that the metabolic activity of purinosomes was signi - alize with the actin network (Fig. 2). However, purinosomes were cantly suppressed in the absence of microtubules. Collectively, we found associated with microtubule filaments in the cytoplasm (Fig. propose a microtubule-assisted mechanism for functional purino- 3). In addition, we examined how purinosome assembly responds to some formation in HeLa cells. the small-molecule inhibitors cytochalasin D and nocodazole, which interfere with actin and microtubule polymerization, respectively. | protein complex | purine biosynthesis The addition of cytochalasin D in living HeLa cells did not have an impact on the distribution of purinosomes (Fig. 4 A and B). How- nzymes synthesizing inosine monophosphate through a de ever, it was apparent that the dissociation of purinosomes occurred novo purine biosynthetic pathway (Fig. 1A) have long been C D E upon the addition of nocodazole (Fig. 4 and ).Thesedatawith CELL BIOLOGY hypothesized to form a multienzyme complex in cells (1–3). Our small molecules are consistent with the cytoskeleton staining experi- investigation of this hypothesis in vivo successfully revealed that ments described above. Collectively, depolymerization of micro- the human de novo purine biosynthetic colocalize in the tubules by nocodazole appears to disfavor the cluster formation of cytoplasm of human cell lines upon purine depletion (Fig. 1 B purinosomes even under purine-deficient conditions. and C) (4, 5). Subsequently, we proposed a subcellular metabolic organization for de novo purine biosynthesis, the “purinosome,” Suppression of Purinosome Activity by Nocodazole. We then estab- fl in cells (4). More importantly, the association and dissociation of lished a cell-based assay to monitor the ux of de novo purine biosynthesis in the presence and the absence of inhibitors. Lawns the purinosome was regulated by changing the purine levels or by 14 manipulating the activity or expression levels of protein kinase of HeLa cells were pulsed with [ C(U)]-glycine, a of CK2 in live cells (4, 5). glycinamide ribonucleotide synthetase at step 2 of de novo pu- rine biosynthesis. Its incorporation into , the rate of Because cytoskeletal structures have been proposed to play an fl important role in the organization of metabolic enzymes (3), we which represents the ux of de novo purine biosynthesis, was determined via acid extraction and ion exchange resin column explored, in this work, whether the purinosome is associated with chromatography. The 14C incorporation into purines was nor- cellular structural elements. For example, glycolytic enzymes in- malized to the total number of cells in the assay, plotted versus cluding aldolase were identified as bound to actin cytoskeleton in – the time after the pulse, and showed its linear incorporation as mammalian and yeast cells (6 8). Interestingly, dynamic alterna- a function of time. tion of actin structures during the cell cycle seems to be correlated Without an inhibitor, the rate of 14C incorporation of glycine with the glycolysis-mediated production of ATP to satisfy an in- into the pool of cellular purines in HeLa cells grown under purine- creased demand for energy (9). Therefore, we sought the struc- depleted conditions was ∼42% greater than the rate observed for tural and functional relationships between the purinosome and cells grown under purine-rich conditions (Fig. 5A). We then cellular cytoskeletal structures using human formylglycinamidine treated cells with the microtubule disrupting agent nocodazole to ribonucleotide synthase (hFGAMS) fused with monomeric green/ assess its effect on the functionality of purinosomes. After a 1-h orange fluorescent proteins (GFP/OFP) as a purinosome marker. incubation with nocodazole, [14C(U)]-glycine was similarly pulsed To visualize or manipulate the cytoskeleton in the presence of for 3 h (Fig. S1). Although purine-rich HeLa cells barely respon- purinosomes, we probed cellular actin networks using rhodamine- ded to nocodazole (Fig. 5B), the flux of de novo purine bio- conjugated phalloidine to stain F-actin structures within fixed cells synthesis for purine-depleted HeLa cells was suppressed by ∼36% and alternatively inhibited actin polymerization by the addition of at the 3-h time point in the presence of nocodazole relative to the cytochalasin D into live cells. In parallel, we stained microtubule DMSO control (Fig. 5B). This experiment clearly showed that filaments in live cells using a TubulinTracker Green reagent purine-deficient cells had diminished de novo purine biosynthesis (Taxol conjugated with Oregon Green 488) and also treated live in the absence of microtubules. Thus, HeLa cells maintained in cells with nocodazole, which directly binds to tubulin so as to in- terfere with microtubule formation in cells. Moreover, we estab- lished a cell-based assay monitoring the flux of de novo purine Author contributions: S.A. and S.J.B. designed research; S.A., Y.D., and J.W.T. performed research; M.K. contributed new reagents/analytic tools; S.A., Y.D., J.W.T., M.K., and S.J.B. biosynthesis to demonstrate the metabolic functionality of puri- analyzed data; and S.A. and S.J.B. wrote the paper. nosomes in the presence and the absence of small molecules. The authors declare no conflict of interest. Collectively, we propose that the spatial distribution of function- 1To whom correspondence should be addressed. E-mail: [email protected]. ally active purinosomes is controlled by the network of micro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tubules when cells demand purine production. 1073/pnas.1008451107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008451107 PNAS Early Edition | 1of5 Downloaded by guest on September 24, 2021 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 carbox- amide ribonucleotide transformylase; ASL, ; CAIRS, carboxyaminoimidazole ribonucleotide synthase; FGAMS, formylglycinamidine ri- bonucleotide 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 , 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.)

purine-depleted conditions rely on the functional clustering of The observed localization phenotype between purinosomes purinosomes that is spatially organized by microtubule networks. and microtubules (Fig. 3) is also supported by our previous observations. When a region of interest containing a purinosome Discussion cluster was photobleached, fluorescent intensities were recovered We conducted cellular imaging of cytoskeletal structures in the in the same location as the photobleached area (4). In addition, in presence of purinosomes. Purinosomes were clearly embedded experiments in which sequential enrichment and depletion of within a network of microtubules, but actin filaments were not purine levels triggered the dissociation and the association of associated or colocalized with purinosomes. In addition, dis- purinosomes (4), reclustering of purinosomes did not occur in the ruption of the microtubule network by the addition of nocoda- same location. Therefore, we propose that newly forming puri- zole was sufficient to dissociate purinosomes in live HeLa cells. nosomes would stochastically nucleate at any location in the cy- The spatial distribution of purinosomes results from their being toplasm guided by microtubules but would remain at that location embedded within microtubule networks. until they functionally dissociate. We then demonstrated that the association and dissociation of As mentioned earlier, glycolytic enzymes are reversibly bound purinosomes were correlated with the rate of de novo purine bio- to actin structures, and glycolysis-driven ATP production was synthesis in HeLa cells. By monitoring 14C-glycine incorporation associated with actin cytoskeleton dynamics (9). In parallel, the into the pool of cellular purines, we were able to observe increased anticipated association of de novo purine biosynthesis with de purine biosynthesis in cells with purinosomes. More importantly, novo ATP synthesis suggests an advantage for the subcellular nocodazole attenuated the metabolic flux of de novo purine bio- localization of purinosomes to microtubules owing to their need synthesis in purine-depleted HeLa cells. Therefore, this cell-based for a ready energy source to perform microtubule-mediated cel- purinosome activity assay indeed supports the role of microtubules lular dynamics. Alternatively, because purinosomes are distrib- for functional purinosome formation in live cells. uted across the entire cytoplasm, microtubule-assisted functional

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,redinC). (Scale bar, 10 μm.)

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1008451107 An et al. Downloaded by guest on September 24, 2021 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,redinC). (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.) CELL BIOLOGY

purinosome formation might be an alternative means of main- were obtained from Sigma. [14C(U)]-Glycine was from DuPont/New En- taining cellular energy homeostasis throughout the cytoplasm. gland Nuclear. Although it is possible that specific microtubule-associated pro- Transfection of Mammalian Cells. teins facilitate purinosome formation, we conclude that the net- A human cervical cancer cell line, HeLa (ATCC), was maintained and transfected for this study as described before (4). work of microtubules minimally provides nucleation sites for Briefly, HeLa cells were subjected to the following: “purine-depleted me- functionally active purinosome formation in the cytoplasm upon dium,” RPMI 1640 (Mediatech) supplemented with dialyzed 5% FBS (Atlanta purine starvation. Biological) and 50 μg/mL gentamicin sulfate (Sigma); and “purine-rich me- dium,” MEM (Mediatech) with 10% FBS and 50 μg/mL gentamicin sulfate. FBS Materials and Methods was dialyzed against 0.9% NaCl at 4 °C for ∼2 d. Lipofectamine 2000 (Invi- Materials. The hFGAMS-GFP and hFGAMS-OFP constructs were prepared as trogen) as a transfection reagent was used by following the manufacturer’s described before (4). Rhodamine-phalloidine and TubulinTracker Green protocol as previously described (4). Of note, an alternative purine-depleted were purchased from Molecular Probes. Cytochalasin D and nocodazole medium (i.e., MEM, dialyzed 10% FBS and 50 μg/mL gentamicin sulfate) was

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 (un- treated; 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.)

An et al. PNAS Early Edition | 3of5 Downloaded by guest on September 24, 2021 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.

evaluated with respect to purinosome formation in HeLa cells by transiently at least three passages. These cells were then seeded into T75 flasks con- expressing hFGAMS-GFP. taining the appropriate, gentamicin-free media at 2 × 106 and 3 × 106 cells/ flask for the purine-rich and purine-depleted conditions, respectively. After Fluorescence Microscopy of Live and Fixed Cells. All samples were imaged at allowing ≈36 h for the cells to achieve midlog phase growth, the cells were ambient temperature (∼25 °C) with a 60× objective (1.49 numeric aperture; placed in 2 mL fresh media. After reequilibration, the cells were pulsed with Nikon Apo TIRF) using a Photometrics CoolSnap ES2 CCD detector mounted [14C(U)]-glycine (125 μM, 20 mCi/mmol, 5 μCi/flask) for the desired time. The onto a Nikon TE-2000E inverted microscope as described before (5). Oregon media was aspirated, and the cells were washed three times with 10 mL ice- Green 488 and GFP detection was accomplished using a S484/15x excitation cold Dulbeccos’s PBS (Cellgro). Cells were harvested by treatment with filter (Chroma Technology), S517/30m emission filter (Chroma Technology), 0.25% trypsin–EDTA solution. and Q505LP/HQ510LP dichroic (Chroma Technology). Rhodamine and OFP To each cell pellet, 1 mL perchloric acid (0.4 M) was added, followed by fi detection was carried out using a S555/25x excitation lter (Chroma Tech- vigorous vortexing to suspend cells. Incubation of cell suspensions in a boiling fi nology), S605/40m emission lter (Chroma Technology) and Q575LP/HQ585LP water bath for 1 h completely lysed the cells and extracted all purines. Im- dichroic (Chroma Technology). mediately after the acid extraction, the tubes were cooled on ice. Cellular Cytochalasin D and nocodazole were added to cells after three washes debris was pelleted by centrifugation and the supernatant was loaded onto with buffered saline solution (20 mM Hepes, pH 7.4, 135 mM NaCl, 5 mM KCl, 0.8 × 3 cm AG50W-X8 (100–200 mesh, Bio-Rad) columns that had been 1 mM MgCl , 1.8 mM CaCl and 5.6 mM glucose). Cells transiently expressing 2 2 preequilibrated with 0.1 M HCl. The columns were washed with 5 mL HCl hFGAMS-GFP were imaged before and after the addition of either 2 μLcy- (1 M), and purines were then eluted with 5 mL of HCl (6 M). Quantitation tochalasin D (1 mg/mL in DMSO) or 4 μL nocodazole (4.2 mg/mL in DMSO) to was achieved by mixing 1 mL of the eluant with 10 mL Ecoscint (National give final concentrations of 1 μg/mL cytochalasin D and 8 μg/mL nocodazole, Diagnostics) followed by liquid scintillation counting using a Beckman respectively. Control experiments were also performed by the addition of Coulter LS6500 instrument. To obtain comparable rates between purine-rich 4 μL DMSO. and purine-depleted conditions, de novo purine biosynthesis was normal- To stain cellular microtubule filaments, live HeLa cells transiently ex- pressing hFGAMS-OFP were washed with buffered saline solution, followed ized to the total number of cells. by incubation with a TubulinTracker Green reagent (1 mM in DMSO; final Effects of Nocodazole on de Novo Purine Biosynthetic Rate. concentration, 250 nM) at 37 °C for 30 min. In addition, to investigate the HeLa cells cultured fl cellular distribution of actin filaments in fixed HeLa cells, cells were prepared in purine-rich and purine-depleted media were inoculated into T75 asks similarly to those used for live cell imaging; however, the cells transfected following the protocol for measuring the de novo purine biosynthetic rate. with hFGAMS-GFP were fixed with freshly prepared 3% formaldehyde, On the day of assay, cells were rinsed and equilibrated in buffered saline permeabilized with 0.2% Triton X-100, and blocked with 10% normal goat solution for 1 h before adding nocodazole (final concentration, 8 μg/mL) or serum (Jackson ImmunoResearch Laboratory) for 30 min at RT as described DMSO as control. After an additional 1 h incubation with nocodazole or before (4). The cells were then incubated for 20 min at RT with rhodamine- DMSO, the cells were pulsed with [14C(U)]-glycine (125 μM, 20 mCi/mmol, μ fl phalloidine (8.3 μM in DMSO; 5 μL/sample) in PBS (PBS: 10 mM Na2HPO4, 5 Ci/ ask) and incubated at 37 °C for 1, 2, and 3 h before harvesting the 14 14 pH 7.4, 2 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl). cells to measure C incorporation into purines as described above. Cin- corporation into newly synthesized purines was normalized to the total Determination of de Novo Purine Biosynthetic Rates. The rate of de novo number of cells. We also performed an unpaired one-tailed Student t test purine synthesis was determined by the incorporation of [14C(U)]-glycine using Microsoft Excel to determine whether the effect of nocodazole on (DuPont/New England Nuclear, NEC-276E, 111.70 mCi/mmol) into cellular purine-depleted HeLa cells was statistically significant. purines using the method of Boss and Erbe (10). HeLa cells were maintained in purine-rich and purine-depleted media (MEM supplemented with 10% ACKNOWLEDGMENTS. This work was funded by National Institutes of FBS or 10% dialyzed FBS, respectively, with 50 μg/mL gentamicin sulfate) for Health Grant GM24129 (to S.J.B.).

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