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Microtubule-Directed Transport of Purine Metabolons Drives Their Cytosolic Transit to Mitochondria

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Microtubule-Directed Transport of Purine Metabolons Drives Their Cytosolic Transit to Mitochondria Microtubule-directed transport of purine metabolons drives their cytosolic transit to mitochondria Chung Yu Chana,b,1, Anthony M. Pedleyb,1, Doory Kimc,1,2, Chenglong Xiac, Xiaowei Zhuangc,d,e,3, and Stephen J. Benkovicb,3 aDepartment of Engineering Science and Mechanics, The Pennsylvania State University, University Park, PA 16802; bDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802; cDepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; dHoward Hughes Medical Institute, Harvard University, Cambridge, MA 02138; and eDepartment of Physics, Harvard University, Cambridge, MA 02138 Edited by David G. Drubin, University of California, Berkeley, California, and accepted by Editorial Board Member Michael R. Botchan November 4, 2018 (received for review August 14, 2018) To meet their purine demand, cells activate the de novo purine bio- In this study, we employed a combination of stochastic optical synthetic pathway and transiently cluster the pathway enzymes into reconstruction microscopy (STORM) (19) and instantaneous metabolons called purinosomes. Recently, we have shown that structured illumination microscopy (VT-iSIM) to visualize the purinosomes were spatially colocalized with mitochondria and localization and movement of purinosomes within the cytosol of microtubules, yet it remained unclear as to what drives these associa- hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient tions and whether a relationship between them exist. Here, we fibroblasts derived from patients diagnosed with Lesch-Nyhan employed superresolution imaging methods to describe purinosome disease (20). These cells rely on the de novo purine biosynthetic transit in the context of subcellular localization. Time-resolved imaging pathway to generate purines and show a twofold enrichment of of purinosomes showed that these assemblies exhibit directed motion purinosome-positive cells compared with a normal, asynchronous as they move along a microtubule toward mitochondria, where fibroblast cell population (20). By using STORM, the average upon colocalization, a change in purinosome motion was observed. diameter and density distributions of purinosomes in Lesch-Nyhan A majority of purinosomes colocalized with mitochondria were also disease are shown to be comparable with those in purine-depleted deemed colocalized with microtubules. Nocodazole-dependent HeLa cells (SI Appendix, Fig. S1). Colocalization analysis of microtubule depolymerization resulted in a loss in the purinosome– FGAMS, our marker for the purinosome, with either mitochondria CELL BIOLOGY mitochondria colocalization, suggesting that the association of puri- or microtubules was performed by 3D STORM (21) and resulted nosomes with mitochondria is facilitated by microtubule-directed in a high degree of colocalization, similar to that of our previous transport, and thereby supporting our notion of an interdepen- studies in purine-depleted HeLa cells (14). Depending on their dency between these subcellular components in maximizing purine colocalization mitochondria or microtubules, different types of production through the de novo purine biosynthetic pathway. motions were revealed for purinosomes through time-lapse im- aging by VT-iSIM. Characterization of directed motions revealed purine metabolism | metabolon | superresolution microscopy | a high tendency for purinosomes to be localized to microtubules mitochondria | cytoskeleton and their motion directed toward mitochondria, suggesting a mechanism by which purinosomes are trafficked to mitochondria n emerging trend in metabolism is that metabolic enzymes via the microtubule network. Disruption of this network resulted Aform supramolecular complexes, called metabolons, to en- in a decrease in purinosome–mitochondria colocalization, hance metabolic flux (1–4). Unlike previously reported metab- olons in the tricarboxylic acid cycle (5) and glycolysis (6), enzymes Significance within the de novo purine biosynthetic pathway assemble into transient, nonmembrane-bound clusters called purinosomes (2, 7, This study draws on the power of superresolution microscopy 8). Proximity assays demonstrated that purinosomes are composed to investigate how metabolons behave near different sub- of core and peripheral proteins and likely assemble in a step-wise cellular components. We revealed an interdependent relation- manner (9, 10). Further characterization of purinosome regulation ship among purinosomes, mitochondria, and microtubules. This revealed that formation is likely mediated through the involvement further suggests a role for each in maximizing purine pro- of molecular chaperones (11, 12) and kinases (13, 14). The degree duction in times of high intracellular demand. With the in- of purinosome assembly is reflected in the cell’s overall intracellular creasing number of reported metabolons, this study has purine demand and serves as a biomarker for pathway activation uncovered a potential general strategy for how metabolons (15). Under cellular conditions that promote purinosome forma- use subcellular networks to facilitate metabolic trade between tion, the metabolic flux through the pathway was shown to be themselves and other cellular organelles. enhanced (16). Recently, imaging studies had revealed colocalization between Author contributions: C.Y.C., X.Z., and S.J.B. designed research; C.Y.C., A.M.P., D.K., and C.X. performed research; C.Y.C., A.M.P., D.K., and C.X. analyzed data; and C.Y.C., A.M.P., purinosomes and subcellular structures (i.e., mitochondria and X.Z., and S.J.B. wrote the paper. microtubules) in HeLa cells, suggesting that the purinosome might The authors declare no conflict of interest. be highly dependent on one or the other for spatial organization This article is a PNAS Direct Submission. D.G.D. is a guest editor invited by the within the cell (14, 17). The de novo process in which the purines Editorial Board. are made is energy-intensive and requires five molecules of ATP, Published under the PNAS license. numerous substrates, and cofactors for every molecule of inosine 1C.Y.C., A.M.P., and D.K. contributed equally to this work. monophosphate generated. Studies have shown that the formate 2Present address: Department of Chemistry, Hanyang University, Seoul, South Korea 133- exported from mitochondria is an essential precursor for the 10- 791. formyltetrahydrofolate cofactor, and elevated production of mi- 3To whom correspondence may be addressed. Email: [email protected] or tochondrial formate results in enhanced metabolic flux through [email protected]. the pathway (18). These observations help support the hypothesis This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that close proximity of purinosomes to mitochondria would be 1073/pnas.1814042115/-/DCSupplemental. advantageous in meeting the catalytic demands of the enzymes. www.pnas.org/cgi/doi/10.1073/pnas.1814042115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 supporting the notion of an interplay between these subcellular Colocalization of Purinosomes with Mitochondria and Microtubules bodies. by Three-Color VT-iSIM. To obtain further evidence that some puri- nosomes are colocalized with both mitochondria and microtubules, Results we performed three-color imaging of live cells, using VT-iSIM (Fig. Colocalization of Purinosomes with Mitochondria and Microtubules 2A). The general localization of purinosomes with mitochon- by Two-Color STORM. Colocalization studies of purinosomes with dria and microtubules is illustrated in the representative region mitochondria in HPRT-deficient fibroblasts were performed of interest (ROI; Fig. 2A, Inset). The image in the ROI is split using two-color 3D STORM imaging of cells transiently expressing into individual channels for better visualization (Fig. 2B). We the purinosome marker FGAMS-mMaple3 and immunostaining found 57.1 ± 13.2% of purinosomes colocalized with mitochondria, ± the mitochondrial outer membrane translocase (TOM20) with the which is statistically greater than the randomization control (22.5 P < C – photoswitchable dye Alexa Fluor 647 (Fig. 1 A and B). The per- 10.2%; 0.05; Fig. 2 ). Substantial purinosome mitochondria centage of purinosomes colocalized with mitochondria was found colocalization was thus observed with both imaging methods, and to be 81.4 ± 6.7%, which was significantly higher than that obtained the quantitative difference in the extent of colocalization observed from the randomized distribution of purinosomes in the cytosol between the two methods may be attributed to differences in the acquisition and image analysis methods, or sample-to-sample varia- (42.9 ± 6.3%; P < 0.05; Fig. 1C and SI Appendix, Supplementary tions. Furthermore, we observed that essentially all mitochondria- Materials and Methods). associated purinosomes were also colocalized with microtubules We also observed a high level of colocalization between puri- (Fig. 2D). Of the remaining purinosomes, the majority (86%) were nosomes (FGAMS-mMaple3) and microtubules immunolabeled also associated with microtubules. These observations invite the with Alexa Fluor 647 (Fig. 1 D and E). Statistical analysis showed – – question as to whether microtubules drive purinosome mitochondria that the purinosome microtubules colocalization percentage was colocalization. 91.6 ± 3.0%, which was significantly higher than that of a ran- domized purinosome distribution (60.5 ± 5.6%; P < 0.05; Fig. 1F). Analysis of Purinosome
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