3. A. Dickinson, Philos. Trans. R. Soc. London Ser. B 308, 67–78(1985).

4. B. W. Balleine, J. P. O’Doherty, Neuropsychopharmacology 35,48–69 (2010).

5. L. H. Corbit, B. C. Chieng, B. W. Balleine,Neuropsychopharmacology 39, 1893–1901 (2014).

6. E. Dias-Ferreira et al., Science 325, 621–625 (2009).7. G. Schoenbaum, B. Setlow, Cereb. Cortex 15, 1162–1169 (2005).8. Supplementary materials are available on Science Online.9. K. D. Ersche et al., Science 335, 601–604 (2012).10. R. Z. Goldstein, N. D. Volkow, Nat. Rev. Neurosci. 12, 652–669

(2011).11. R. J. Beninger, S. T. Mason, A. G. Phillips, H. C. Fibiger,

J. Pharmacol. Exp. Ther. 213, 623–627 (1980).12. J. D. Salamone, M. Correa, Behav. Brain Res. 137, 3–25

(2002).13. J. W. Dalley et al., Science 315, 1267–1270 (2007).14. D. Belin, A. C. Mar, J. W. Dalley, T. W. Robbins, B. J. Everitt,

Science 320, 1352–1355 (2008).15. N. D. Volkow et al., Nature 386, 830–833 (1997).16. D. Martinez et al., Am. J. Psychiatry 166, 1170–1177 (2009).

17. M. J. Frank, L. C. Seeberger, R. C. O’Reilly, Science 306,1940–1943 (2004).

18. S. de Wit et al., Psychopharmacology 219, 621–631 (2012).19. K. Wunderlich, P. Smittenaar, R. J. Dolan, Neuron 75, 418–424

(2012).20. Z. Sjoerds et al., Transl. Psychiatry 3, e337 (2013).21. J. M. Barker, J. R. Taylor, Neurosci. Biobehav. Rev. 47, 281–294

(2014).22. C. M. Gillan et al., Am. J. Psychiatry 168, 718–726 (2011).23. C. M. Gillan, M. Kosinski, R. Whelan, E. A. Phelps, N. D. Daw,

eLife 5, e11305 (2016).24. C. M. Gillan et al., Biol. Psychiatry 75, 631–638 (2014).

ACKNOWLEDGMENTS

We thank all volunteers for their participation in this study, as wellas the staff at the Mental Health Research Network and theCambridge BioResource for their assistance with volunteerrecruitment. We are especially grateful to N. Flake and S. Whittlefor their exceptional commitment in this regard. We also thank thestaff at the National Institute for Health Research (NIHR) ClinicalResearch Facility at Addenbrooke’s Hospital for their support

throughout this study. We are grateful to S. Abbott, R. Lumsden,J. Arlt, C. Whitelock, I. Lee, and M. Pollard for their assistance.C.M.G. is supported by a Sir Henry Wellcome PostdoctoralFellowship (101521/Z/12/Z). This work was funded by a grant fromthe Medical Research Council (MR/J012084/1) and was conductedwithin the NIHR Cambridge Biomedical Research Centre and theBehavioral and Clinical Neuroscience Institute, which is jointlyfunded by the Medical Research Council and the WellcomeTrust. The data described in this paper are stored at theUniversity of Cambridge's institutional repository, Apollo(http://dx.doi.org/10.17863/CAM.122).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6292/1468/suppl/DC1Materials and MethodsSupplementary TextTables S1 and S2References (25–30)

3 February 2016; accepted 19 May 201610.1126/science.aaf3700

STEM CELLS

Spatiotemporal coordination ofstem cell commitment duringepidermal homeostasisPanteleimon Rompolas,1*† Kailin R. Mesa,1† Kyogo Kawaguchi,2 Sangbum Park,1

David Gonzalez,1 Samara Brown,1 Jonathan Boucher,1

Allon M. Klein,2‡ Valentina Greco1,3‡

Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cellself-renewal, commitment, and functional integration into the tissue remain unsolved.Using imaging techniques in live mice, we captured the lifetime of individual cells in the earand paw epidermis. Our data suggest that epidermal stem cells have equal potential toeither divide or directly differentiate. Tracking stem cells over multiple generations revealsthat cell behavior is not coordinated between generations. However, sibling cell fate andlifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, wedemonstrated that differentiating stem cells integrate into preexisting ordered spatialunits of the epidermis. This study elucidates how a tissue is maintained by both temporaland spatial coordination of stem cell behaviors.

Tissue homeostasis requires the ability to re-place damaged or lost cells while maintain-ing tissue structure and function. A modelfor studying this process is themouse adultinterfollicular epidermis (IFE), where orga-

nized layers of progressively differentiated epi-thelial cells form a barrier fromwhich suprabasalcells are continuously shed and replenished byanunderlyingproliferativebasal layer (1–3).Under-standing how basal stem cell proliferation and

terminal differentiation remain balanced in ho-meostasis is a central question in both epithelialand stem cell biology.Initial models of epidermal maintenance re-

cognized the three-dimensional organization ofdiscrete columns, called epidermal proliferativeunits (EPUs), which are defined by the perimeterof the most external, terminally differentiatedcells (4–6). An important implication of the EPUmodel is that each unit is autonomously main-tained by an asymmetrically dividing, basally lo-cated stem cell, with slow-cycling characteristics(7–9). Recent studies support the presence of slow-cycling stem cells in mouse epidermis (10, 11).However, long-term lineage-tracing studies showthat basal clones do not strictly adhere to thecolumnar borders of EPUs and support a modelbased on a single stem cell population that makesstochastic fate choices, while still relying onmost-ly (60 to 84%) asymmetric divisions to generateone stem cell and one terminally differentiated

cell (12–16). These studies provide critical insightsinto epidermal homeostasis but remain discon-nected and don’t explain how individual stemcells and their progeny are integrated into theexisting structure of a tissue.A major challenge in elucidating cell fate has

been the inability to resolve individual cell fatechoices within clones. Individual cell behaviorshave been indirectly inferred from time series offixed clonal samples (17). Therefore, we developedan in vivo pulse/chase system for single-cell genet-ic label retention to continuously track entire lin-eages across multiple generations and capturethe fate of individual basal cells within them(18) (Fig. 1A and fig. S1A). For that, we acquiredserial optical sections of the epidermis from thesame live adult mice at successive time pointsand captured the differentiation state of singlelabeled cells by position and cellular morphol-ogy within the entire volume of the IFE (fig. S1B)(19–22). To distinguish between region-specificcharacteristics and more general epidermal prin-ciples, we performed our lineage tracing in bothear and plantar epidermis. Cells that committedto differentiation were scored by their departurefrom the basal layer and their gradual movementtoward the surface of the skin, which was ir-reversible in all cases (fig. S1C). Cell divisions inthe basal layer generated two daughter cells thatremained within the basal layer upon division(fig. S1D).Analysis of division and differentiation events

in clonal lineage trees provided direct access tolifetimes and fate choices of individual basal cells,and revealed fate correlations that could not beaddressed from static clonal analysis (Fig. 1, Band C, and fig. S2A). We tested two key hypothe-ses: First, we asked whether the basal layer ismaintained through a proliferative hierarchy bya small population of stem cells (10, 11); if so,mother and daughter cell fates should be cor-related, because only stem cells should give riseto daughter stem cells. We performed this mul-tigenerational analysis in the ear epidermis anddetected no mother-daughter bias in fate choice[supplementary theory (ST) S5] or in their life-times (Pearson correlation R = –0.11, P = 0.2).

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1Department of Genetics, Yale School of Medicine, NewHaven, CT 06510, USA. 2Department of Systems Biology,Harvard Medical School, Boston, MA 02115, USA.3Departments of Dermatology and Cell Biology, Yale StemCell Center, Yale Cancer Center, Yale School of Medicine,New Haven, CT 06510, USA.*Present address: Department of Dermatology, Institute forRegenerative Medicine, University of Pennsylvania PerelmanSchool of Medicine, Philadelphia, PA 19104, USA.†These authors contributed equally to this work. ‡Correspondingauthor. Email: valentina.greco@yale.edu (V.G.); allon_klein@hms.harvard.edu (A.M.K.)

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Second, we tested whether asymmetric fate di-visions are the main mode of self-renewal, aswidely suggested from static lineage tracing(10, 11, 15, 23). Asymmetric divisions should re-sult in anticorrelated sister cell fates, but wefound that sister cell fates were either indepen-dent (ear) or positively correlated (paw). In bothtissues, we found that sister cells had stronglycorrelated lifetimes (Fig. 2, A and B, and ST S4).Such sibling correlations could be indicative ofcoupled activities due to spatial co-localization orco-inheritance.These results suggest a simple model for stem

cell fate without hierarchy or division asymmetry(23) (Fig. 2, B and C; figs. S2, B to E, and S3; andSTs S2 and S3). This model excludes fate asym-metry, does not imply fate commitment at birth,and demonstrates the temporal and fate coordi-nation of adult sister stem cell behaviors in vivo.Todate, ourwork, alongwith the field in general,

has relied on genetic approaches based on Cre-recombinase lineage tracing, which could poten-tially introduce bias (10, 11, 24). We thereforeused two additional independent lineage-tracingapproaches to track single cells and populationswithin the epidermis, respectively. First, we en-gineered a transgenic mouse that expresses alight-activatable fluorescent reporter (26) in theepidermis (fig. S4 and movie S1). Using two-photon illumination to randomly label and trackindividual basal cells, we found that basal cell be-havioral kinetics are comparable to those shownby our Cre lineage-tracing analysis, supportingour proposed stem cell model (Fig. 3, A and B,and fig. S5). Furthermore, we labeled geomet-rically defined regions within different epider-mal layers to track the uniformity of populationcell fate over time (movie S2). By these means,we found a global basal layer turnover as wellas rapid progression of committed cells throughthe suprabasal layers within only a few days(Fig. 3C). Second, we used apreviouslydevelopedtetracycline-inducible H2B-GFP (GFP, green fluo-rescent protein) label retention system (10, 11, 18)to track cells for up to 2 weeks in the ear epider-mis and found global dilution of the H2B-GFPsignal already by 1 week. No cells (n = 11,370)

retained their GFP label, even after correctingfor background signal decay, suggesting that allbasal cells cycle at similar rates (fig. S6, A to F).Collectively, our work has demonstrated thatthe basal layer of the epidermis is composed ofa single equipotent stem cell population, throughthe utilization of three powerful and complemen-tary label retention approaches in live mice.We next sought to understand how these seem-

ingly random cell fate decisions in the basal layercontribute to organized suprabasal differentiatedlayers. Previous work has demonstrated that cellsin the basal layer are not constrained to columnboundaries, and it has been proposed that cellscould differentiate through self-assembling pro-

cesses (23, 27, 28). To interrogate the mechanismby which cells transit through suprabasal layers,we used a dual membrane fluorescent reporterto differentially label neighboring cells in the earepidermis (fig. S1).We find that neighboring cellsindependently transit through the differentiatedepidermal layers before they are shed from theskin (Fig. 4A). This suggests that within layers,the lateral connections between cells are dynamicduring differentiation. Analysis of cell bordersshowed that the global organization in the ter-minally differentiated granular layer remainedrelatively unchanged in the short term (fig. S8A).To reconcile the highly dynamic reorganization

of the basal layer with the seemingly unchanged

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Fig. 1. Subclonal lineage tracingof basal epidermal cells.(A) Experimental approach for epi-dermal fate tracking by single-celllabel retention. After clonalinduction with tamoxifen,K14CreER ;R26flox-stop-tTA/mTmG;pTREH2BGFPmice were treatedwith doxycycline to evaluateH2BGFPlabel retention. (B) The fate andsubsequent behavior of identifiedcells were determined by live imagingat 2-day intervals.The epidermis iscomposed of cellular layers startingfrom themost external, cornified layer,then moving inward to the granular,spinous, and finally basal layer. The dashed outlines indicate the boundaries of the traced clone. (B) and (C) Representative time sequence of a single-cell labelretention experiment, showing the resulting lineage tree (C),with individual cell fate choices identified through label dilution within the same clone. Scale bar, 25 mm.

Day 0 Day 2 Day 4 Day 6

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Fig. 2. Basal stem cells make stochastic fate choices that are temporally coordinated. (A) Theproportion of divisions leading to symmetric and asymmetric fates, and the magnitude and significanceof sister cell lifetime correlations, measured directly from lineage trees (n= 136 divisions across n = 40 treesin the ear, and n =101 divisions across n = 92 trees in the paw). Color shows the statistical significance ofcorrelations: P > 0.05 (blue), P < 10−4 (red). (B) A stochastic model of cell fate, with each cell dividing ordirectly differentiating after a minimum refractory period, with a fluctuating division probability P balancedat 50% in homeostasis. Spatial or lineage-coupled fluctuations in P between sister cells, measured by thevariance in P, lead to correlated sister cell fates. t, average cell lifetime, g, stochastic division/differentiationrate after a refractory period.The model is mathematically defined in ST S3. (C) A fit of the model to thedistribution of clone sizes (basal cells per clone) over time (n = 40 clones); error bars, SEM.

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architecture of the terminally differentiated layers,we examined the trajectory of individual cells fromthe basal to the cornified layers. We find that themajority of committed cells align into vertical col-umns, giving rise to the structures that inspiredthe EPU hypothesis (29) (figs. S7 and S8). There-fore, we propose that such columns are epidermaldifferentiation units (EDUs), rather than EPUs.

These columns persist over time as newly differ-entiated cells move upward to recycle the samespace occupied by preceding cells, but they arenot entirely stable because a minority of cells(~10%) do not funnel into preexisting EDUs butinstead form new units as they transit throughthe layers of the epidermis (Fig. 4, C and D). Fur-thermore, our lineage-tracing data show that

although cells inhabiting the basal and spinouslayers have the flexibility to switch to neighbor-ing EDUs as cells arrive in the granular layer,their fate is vertically fixed into a supply chain forthe cornified layers above (Fig. 4E). Analysis ofthe distribution of residence times of cells in thesuprabasal layers shows that cell maturation inthe spinous layer spans the same typical time foralmost all cells, and their subsequent exit fromthe granular layer appears to be stochastic (STS2). This suggests that after departure from thebasal layer, the granular layer spatially coordi-nates the transition to cornified tissue by actingas a buffer zone. Furthermore, the identificationof a small percentage of cells that are capable ofcreating new differentiation paths suggests amechanism to confer flexibility for epidermal re-modeling over time (Fig. 4E).These results support a simple model of epi-

dermal maintenance proposed over 50 yearsago (18, 24), in which basal cells are born as un-committed stem cells, with an equal chance toultimately proliferate or differentiate. Siblingstem cells coordinate fate commitment and tem-poral execution of their behaviors. Finally, ascells depart from the basal layer, they funnel pre-dominantly into metastable EDUs. Taken togeth-er, this study demonstrates how spatiotemporalcoordination in both the proliferative and differ-entiated layers sustains epidermal maintenanceand function. This provides a foundation for futurework to investigate the largely unknown regulatorymechanisms that coordinate cell-cell interactionson a tissue scale, during homeostasis. Furthermore,such progressive acquisition of cell fate couldallow for cells in the epidermis to remain flexiblein response to environmental demands, as wasrecently suggested from work on human keratin-ocytes in vitro (30), and therefore has potentialrelevance to pathological states and regeneration.

REFERENCES AND NOTES

1. V. Levy, C. Lindon, B. D. Harfe, B. A. Morgan, Dev. Cell 9,855–861 (2005).

2. E. Christophers, E. B. Laurence, Curr. Probl. Dermatol. 6,87–106 (1976).

3. M. Ito et al., Nat. Med. 11, 1351–1354 (2005).4. C. Potten, Cell Prolif. 7, 77–88 (1974).5. S. Ghazizadeh, L. B. Taichman, EMBO J. 20, 1215–1222

(2001).6. I. C. Mackenzie, J. Invest. Dermatol. 109, 377–383 (1997).

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Cel

ls tr

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(%

)

Pre-existing New shape

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100

Basal Spinous Granular0

20

40

60

80

100

Cel

ls tr

aced

(%

)

Same Switch

Bas

alG

ranu

lar

Day 0 Day 2 Day 4

New unitUnit switchUnit

mTH2BGFP

Day 0 Day 1 Day 2 Day 3

12 2

3 3 3

4

5 5

6

12

1

3

21

Gra

nula

r

55

4 4 4

666

Fig. 4. Basal cells transit into preexisting epidermal differentiation units. (A) Optical section of asingle plane of the top granular layer of the epidermis, taken over 3 days, depicting individually labeleddifferentiating cells as they transit through at different time points. The majority of differentiating cellsarrive at the same space as their predecessors (yellow arrows), as indicated by the unchanging cellboundaries. (B) Quantification of epidermal differentiation behaviors (n = 40 cells); error bars representSD. (C) Representative examples of differentiating epidermal cells switching or integrating into existingunits (green and yellow arrows, respectively). (D) Quantification of the frequency of unit switching foreach epidermal layer (n = 40 cells); Error bars represent SD. (E) Schematic for epidermal homeostasis.Basal stem cells stochastically commit to differentiation and transit through the suprabasal layers bypredominately using existing columnar units. Scale bars, 25 mm.

Day 0 Day 1

Bas

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pino

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Divided

Differentiated

K14-H2BPAmCherryK14-actinGFP

Divided Differentiated0

1020304050

Photolabeled K14CreER

2-Day cell tracing

Cel

ls tr

aced

(%

)

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rB

asal

Day 0 Day 3

Hair shaft

Hair shafts

K14-H2BPAmCherry

Fig. 3. Unbiased epidermal fate tracking by single-cell photolabeling.(A) Representative examples of cell division and differentiation fates. (B) Quan-tification of cell division and differentiation events. (C) Representative time se-quence of a region labeled with a photoactivatable reporter. At day 0, twoadjacent square areas were scanned to activate the H2BPAmCherry reporter inthe granular and basal layer, respectively. Scale bars, 25 mm.

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7. C. S. Potten, R. J. Morris, J. Cell Sci. Suppl. 1988 (suppl. 10),45–62 (1988).

8. S. Ghazizadeh, L. B. Taichman, J. Invest. Dermatol. 124,367–372 (2005).

9. U. B. Jensen, S. Lowell, F. M. Watt, Development 126,2409–2418 (1999).

10. G. Mascré et al., Nature 489, 257–262 (2012).11. A. Sada et al., Nat. Cell Biol. 18, 619–631 (2016).12. E. Clayton et al., Nature 446, 185–189 (2007).13. A. M. Klein, T. Nakagawa, R. Ichikawa, S. Yoshida, B. D. Simons,

Cell Stem Cell 7, 214–224 (2010).14. D. P. Doupé et al., Science 337, 1091–1093 (2012).15. X. Lim et al., Science 342, 1226–1230 (2013).16. A. Füllgrabe et al., Stem Cell Rep. 5, 843–855 (2015).17. D. P. Doupé, A. M. Klein, B. D. Simons, P. H. Jones, Dev. Cell 18,

317–323 (2010).18. A. M. Klein, B. D. Simons, Development 138, 3103–3111

(2011).19. T. Tumbar et al., Science 303, 359–363 (2004).20. P. Rompolas et al., Nature 487, 496–499 (2012).21. P. Rompolas, K. R. Mesa, V. Greco, Nature 502, 513–518

(2013).22. K. R. Mesa et al., Nature 522, 94–97 (2015).

23. C. M. Pineda et al., Nat. Protoc. 10, 1116–1130 (2015).24. J. P. Marques-Pereira, C. P. Leblond, Am. J. Anat. 117, 73–87

(1965).25. K. Kretzschmar, F. M. Watt, Cell 148, 33–45 (2012).26. F. V. Subach et al., Nat. Methods 6, 153–159

(2009).27. H. Honda, M. Tanemura, S. Imayama, J. Invest. Dermatol. 106,

312–315 (1996).28. D. N. Menton, J. Invest. Dermatol. 66, 283–291

(1976).29. C. S. Potten, J. Invest. Dermatol. 65, 488–500

(1975).30. A. Roshan et al., Nat. Cell Biol. 18, 145–156 (2016).

ACKNOWLEDGMENTS

We thank E. Fuchs for K14-actinGFP mice; V. Verkhusha for thePAmCherry construct; and M. Theo, S. Ghazizadeh, and P. Jonesfor critical manuscript feedback. This work was supported byThe New York Stem Cell Foundation; the Edward Mallinckrodt Jr.Foundation; the National Institute of Arthritis and Musculoskeletaland Skin Disease (NIAMS), grants NIH-5R01AR063663-04 andNIH-1R01AR067755-01A1; and the NIH Predoctoral Program inCellular and Molecular Biology (K.R.M. and S.B., grant

T32GM007223). K.R.M is an NSF Graduate Research Fellow. S.P.is supported by CT Stem Cell grant 14-SCA-YALE-05. P.R. issupported by an American Skin Association Research ScholarAward, was a New York Stem Cell Foundation-DruckenmillerFellow, and was supported by CT Stem Cell grant 13-SCA-YALE-20.V.G. is a New York Stem Cell Foundation Robertson Investigator.A.M.K. is supported by a Career Award at the ScientificInterface from the Burroughs Wellcome Fund and an EdwardMallinckrodt Jr. Foundation grant. The K14-H2BPAmCherrymouse is available from the Greco lab under a materialstransfer agreement with Yale.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6292/1471/suppl/DC1Materials and MethodsAuthor ContributionsSupplementary TextFigs. S1 to S8Movies S1 and S2

16 March 2016; accepted 18 May 2016Published online 26 May 201610.1126/science.aaf7012

METABOLISM

Biosensor reveals multiple sourcesfor mitochondrial NAD+

Xiaolu A. Cambronne,1* Melissa L. Stewart,1 DongHo Kim,1 Amber M. Jones-Brunette,2

Rory K. Morgan,3 David L. Farrens,2 Michael S. Cohen,3* Richard H. Goodman1

Nicotinamide adenine dinucleotide (NAD+) is an essential substrate for sirtuins andpoly(adenosine diphosphate–ribose) polymerases (PARPs), which are NAD+-consumingenzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD+

concentrations within these subcellular compartments are thought to regulate theactivity of NAD+-consuming enzymes; however, the challenge in measuringcompartmentalized NAD+ in cells has precluded direct evidence for this type ofregulation.We describe the development of a genetically encoded fluorescent biosensorfor directly monitoring free NAD+ concentrations in subcellular compartments. Wefound that the concentrations of free NAD+ in the nucleus, cytoplasm, and mitochondriaapproximate the Michaelis constants for sirtuins and PARPs in their respectivecompartments. Systematic depletion of enzymes that catalyze the final step of NAD+

biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD+

concentrations.

Beyond its role in redox reactions, nico-tinamide adenine dinucleotide (NAD+) isan essential substrate for sirtuins andpoly(adenosine diphosphate–ribose) poly-merases (PARPs) (1). Although these enzymes

serve different functions—sirtuins catalyze pro-tein deacylation, whereas PARPs catalyze ADPribosylation—both cleave the glycosidic bondbetween nicotinamide and ADP-ribose, resultingin the irreversible consumption of NAD+ (2, 3).As a consequence, mammalian cells rely on sal-vage pathways that recycle the nicotinamide

generated by these NAD+-consuming enzymes tomaintain NAD+ concentrations above a criticalthreshold. Nicotinamide phosphoribosyltrans-ferase (NAMPT), the enzyme that converts nico-tinamide to nicotinamidemononucleotide (NMN),is essential for maintaining NAD+ concentrations(4). The conversion of NMN to NAD+ is catalyzedby three enzyme isoforms—namely, NMN adenyl-transferases (NMNATs) that are differentiallylocalized (NMNAT1, nucleus; NMNAT2, Golgi,cytosol-facing; NMNAT3, mitochondria) (5)—suggesting the existence of distinct subcellularNAD+ pools. Localized fluctuations in NAD+

levels may regulate the activity of the NAD+-consuming enzymes, which are also highly com-partmentalized (6–8); however, there has beenno direct experimental evidence for this typeof regulation because the concentration of freeNAD+ (i.e., NAD+ available as a substrate) withinthese subcellular compartments has so far been

unknown. Moreover, it has been unclear whetherthese NAD+ pools are segregated or readily ex-changeable between subcellular compartments.To address these issues, we developed a ge-

netically encoded biosensor (9) for measuringfreeNAD+ concentrationswithin subcellular com-partments. This sensor comprises a circularlypermuted Venus fluorescent protein (cpVenus)and a bipartite NAD+-binding domain modeledfrom bacterial DNA ligase (Fig. 1A and fig. S1)that exclusively uses NAD+ as a substrate (10).Point mutations were introduced to preventNAD+ consumption and allow monitoring ofNAD+ within the predicted physiological range.Purified sensor and cpVenus (fig. S2) had majorexcitation peaks at ~500 nm that fluoresced at~520 nm (Fig. 1B). NAD+ decreased sensor fluo-rescence (excitation at 488 nm) in a dose-dependent manner and minimally affectedcpVenus fluorescence (Fig. 1, B and C). A secondexcitation peak at 405 nm was unaffected byNAD+ binding (Fig. 1C and fig. S3), allowingratiometric (488 nm/405 nm) measurements fornormalizing sensor expression levels (fig. S4).In vitro, the apparent NAD+ dissociation con-stant (Kd) of the sensor was ~65 mM (Fig. 1D).Absorbance measurements revealed two ma-jor species at ~415 and ~488 nm that appearedto interconvert upon NAD+ addition around a~450-nm isosbestic point (fig. S5). This indi-cates that the NAD+-bound species loses its fluo-rescence at 488 nm, converting to a species thatabsorbs at 415 nm but is nonfluorescent. Ac-cordingly, NAD+ did not affect the fluorescencelifetime after 488-nm excitation (fig. S6), provid-ing further evidence that fluorescence after488-nm excitation solely represents the unboundfraction.To confirm the reversibility of NAD+ binding,

we showed that elution of NAD+ from the sen-sor returned fluorescence to that of a controlsample (Fig. 1E). We also monitored fluorescencein real time in the presence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which hasa higher affinity for NAD+ than the sensor does

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1Vollum Institute, Oregon Health & Science University,Portland, OR 97239, USA. 2Department of Biochemistry andMolecular Biology, Oregon Health & Science University,Portland, OR 97239, USA. 3Department of Physiology andPharmacology, Oregon Health & Science University, Portland,OR 97239, USA.*Corresponding author. Email: ang@ohsu.edu (X.A.C.); cohenmic@ohsu.edu (M.S.C.)

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Spatiotemporal coordination of stem cell commitment during epidermal homeostasis

Boucher, Allon M. Klein and Valentina GrecoPanteleimon Rompolas, Kailin R. Mesa, Kyogo Kawaguchi, Sangbum Park, David Gonzalez, Samara Brown, Jonathan

originally published online May 26, 2016DOI: 10.1126/science.aaf7012 (6292), 1471-1474.352Science 

, this issue p. 1471Sciencereused the existing spatial organization of the epidermis.previously thought, but through the coordination of sibling cell fate and lifetimes. Furthermore, differentiating stem cells generations revealed that tissue homeostasis in the mouse epidermis is not maintained by asymmetric cell division asvisualized individual stem cells over their lifetime in the epidermis of live mice. Tracking stem cells over multiple

et al.After injury and during homeostasis, tissues rely on the balance of cell loss and renewal. Rompolas Tracking stem cell fate in time and space

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