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Molecular organization of the desmosome as revealed by directstochastic optical reconstruction microscopySara N. Stahley1, Emily I. Bartle1, Claire E. Atkinson2, Andrew P. Kowalczyk1,3 and Alexa L. Mattheyses1,*

ABSTRACTDesmosomes aremacromolecular junctions responsible for providingstrong cell–cell adhesion. Because of their size and molecularcomplexity, the precise ultrastructural organization of desmosomesis challenging to study. Here, we used direct stochastic opticalreconstruction microscopy (dSTORM) to resolve individual plaquepairs for inner and outer dense plaque proteins. Analysis methodsbased on desmosomal mirror symmetry were developed to measureplaque-to-plaque distances and create an integrated map. Wequantified the organization of desmoglein 3, plakoglobin anddesmoplakin (N-terminal, rod and C-terminal domains) in primaryhuman keratinocytes. Longer desmosome lengths correlated withincreasing plaque-to-plaque distance, suggesting that desmoplakin isarranged with its long axis at an angle within the plaque. Wenext examined whether plaque organization changed in differentadhesive states. Plaque-to-plaque distance for the desmoplakinrod and C-terminal domains decreased in PKP-1-mediatedhyperadhesive desmosomes, suggesting that protein reorganizationcorrelates with function. Finally, in human epidermis we found adifference in plaque-to-plaque distance for the desmoplakin C-terminal domain, but not the desmoplakin rod domain orplakoglobin, between basal and suprabasal cells. Our data revealthe molecular organization of desmosomes in cultured keratinocytesand skin as defined by dSTORM.

KEY WORDS: Cell adhesion, Keratinocyte, Super-resolution,Fluorescence, Microscopy, Cadherin, dSTORM

INTRODUCTIONDesmosomes are intercellular junctions that are abundant in tissuesthat experience considerable mechanical stress, such as the skinand heart (Kowalczyk and Green, 2013). The importance ofdesmosome-mediated adhesion is evidenced by the numeroushuman diseases that result when desmosomes are compromised,manifesting in epidermal fragility, cardiomyopathies and cancers(Broussard et al., 2015; Stahley and Kowalczyk, 2015).The desmosome is a macromolecular complex that forms a ‘spot-

weld’ of strong intercellular adhesion and comprises transmembranecadherins and cytoplasmic plaque proteins (Berika and Garrod,2014; Saito et al., 2012). The desmosomal cadherins (desmogleinsand desmocollins) link neighboring cells through extracellularadhesive interactions whereas the armadillo protein family members

plakoglobin and plakophilin, and the plakin family memberdesmoplakin contribute to the intracellular plaque (Fig. 1A).Plaque ultrastructure is characterized by two electron-denseregions: the plasma-membrane-proximal outer dense plaque andthe inner dense plaque (Desai et al., 2009; Farquhar and Palade,1963; Stokes, 2007). The cadherin cytoplasmic tails bind to proteinsin the outer dense plaque whereas the C-terminus of desmoplakinbinds to intermediate filaments in the inner dense plaque. Thistethers the desmosome to the intermediate filament cytoskeleton,establishing an integrated adhesive network (Bornslaeger et al.,1996; Harmon and Green, 2013).

Many desmosomal protein interactions have been characterizedby biochemical studies (Bass-Zubek and Green, 2007; Green andSimpson, 2007; Thomason et al., 2010), and desmosomalultrastructure has been studied by immunogold and tomographicelectron microscopy (Al-Amoudi et al., 2011; Al-Amoudi andFrangakis, 2008; North et al., 1999; Owen et al., 2008; Stokes,2007). Yet, studying desmosomes on a molecular level remainschallenging due to their size, insolubility and molecular complexity.Measuring desmosomal protein organization requires a techniquewith high resolution coupled with minimal sample manipulation tominimize structural artifacts and preserve physiological relevance.Super-resolution microscopy has revealed sub-resolution proteinorganization in numerous macromolecular complexes including thenuclear pore, ciliary transition zone, kinetochore, neuronal synapse,focal adhesion and hemidesmosome (Chatel et al., 2012;Loschberger et al., 2014, 2012; Nahidiazar et al., 2015; Ribeiroet al., 2010; van Hoorn et al., 2014; Yang et al., 2015; Zhong, 2015).Here, we use direct stochastic optical reconstruction microscopy(dSTORM) (Rust et al., 2006) to elucidate protein organizationwithin the desmosome at the molecular level both in vitro andin vivo.

RESULTS AND DISCUSSIONDesmosome plaques resolved by dSTORMTwo proteins, the inner dense plaque protein desmoplakin and theouter dense plaque protein plakoglobin, were utilized to establishdSTORM for studying desmosome organization. Primary humankeratinocytes were pre-extracted prior to fixation and thedesmosomal pool of proteins (Palka and Green, 1997) wasimmunostained with primary antibodies against the desmoplakinC-terminal or plakoglobin N-terminal domains. The same cell–cellborder was then imaged by widefield, structured illuminationmicroscopy (SIM) and dSTORM.

In widefield, desmoplakin fluorescence was punctate andpixilated with no discernable structural features. In contrast, twodistinct desmoplakin plaques, one contributed by each cell, wereresolved by SIM as previously shown (Fig. 1B,C) (Stahley et al.,2015). Likewise, two individual desmoplakin plaques were resolvedby dSTORM, but with a more distinct separation (Fig. 1C).To analyze the difference in spatial resolution between the imagingReceived 7 January 2016; Accepted 15 June 2016

1Department of Cell Biology, Emory University School of Medicine, Atlanta, GA30322, USA. 2Department of Biochemistry and Molecular Biology, University ofChicago, Chicago, IL 60637, USA. 3Department of Dermatology, Emory UniversitySchool of Medicine, Atlanta, GA 30322, USA.

*Author for correspondence (mattheyses@emory.edu)

A.L.M., 0000-0002-5119-7750

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techniques, fluorescence intensity was plotted as a function ofdistance along the desmosome axis, perpendicular to theplasma membrane. Although there is only a single intensity peakfrom the widefield image, two individual peaks, corresponding tothe two plaques, are resolved from the SIM and dSTORM images(Fig. 1D).Plakoglobin-labeled desmosomes imaged by widefield have a

punctate appearance, similar to desmoplakin (Fig. 1E,F). Similarly,plakoglobin fluorescence by SIM is punctate with no evidentinternal structure. In contrast, dSTORM revealed two individual

plakoglobin plaques (Fig. 1F). This is also shown in the plot offluorescence intensity along the desmosome axis (Fig. 1G).

To confirm dSTORM resolves individual desmosomes consistingof plaques contributed by two different cells, keratinocytes weredual-labeled for the extracellular domain of desmoglein 3 (Dsg3)and the C-terminal domain of desmoplakin. dSTORM revealed adistinct separation, with the Dsg3 extracellular domain sandwichedbetween two desmoplakin plaques (Fig. 1H). In our experiments,the localization precision of Alexa Fluor 488 (∼19.5 nm) was lessthan that of Alexa Fluor 647 (∼5.8 nm), similar to that reported by

Fig. 1. Super-resolution imaging of desmosomes. (A) Desmosome schematic. (B,C,E,F) Cultured human keratinocytes labeled for the desmoplakin (DP)C-terminal domain or plakoglobin (PG) N-terminal domain and imaged by widefield microscopy (WF), SIM and dSTORM. (B) SIM image of desmoplakin cellborder. (C) Desmoplakin-labeled region of interest imaged by widefield microscopy, SIM and dSTORM. (D) Linescan across a single desmosome indicated by thecolored boxes. (E) SIM image of plakoglobin cell border. (F) Plakoglobin-labeled region of interest imaged by widefield microscopy, SIM and dSTORM.(G) Linescan across a single desmosome indicated by the colored boxes. (H) dSTORM image of keratinocyte cell–cell border labeled for the Dsg3 N-terminal(Alexa Fluor 488, green) and desmoplakin C-terminal (Alexa Fluor 647, red) domains. Scale bars: 10 µm (B,E); 2 µm (C,F,H); 500 nm (C,F,H, insets).

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others (Dempsey et al., 2011; Tam et al., 2014). Therefore, AlexaFluor 647 was used for quantitative measurements unless otherwiseindicated. Image resolution is also affected by the primary andsecondary antibody labels, which increases the apparent size ofstructures (Bates et al., 2007).In summary, these results demonstrate that dSTORM can resolve

proteins in both the outer and inner dense plaques in individualdesmosomes.

Molecular map of the desmosome by dSTORMNext, we systematically quantified the molecular arrangement ofproteins in the desmosome. Keratinocytes were labeled withantibodies against the Dsg3 N-terminal (Alexa Fluor 488),plakoglobin N-terminal, Dsg3 C-terminal, desmoplakin N-terminal, desmoplakin rod, and desmoplakin C-terminal domains(Fig. 2A; Table S1) and protein distributions along the desmosomalaxis were determined using dSTORM. The mirror symmetry ofdesmosomal plaques allowed us to identify individual desmosomes,and only those with their axes in the x-y focal plane were analyzed(Fig. 2B; Fig. S1). A linescan of fluorescence intensity alongthe desmosome axis was created for each desmosome (Fig. 2C). Theaxial distribution of proteins was automatically measured as thedistance between peaks from the linescans. This plaque-to-plaquedistance was measured from multiple individual desmosomeslabeled for each protein domain (Fig. 2D). There was aprogressive increase in the average plaque-to-plaque distance withthe plakoglobin N-terminal domain closest to the plasma membraneand the desmoplakin C-terminal domain furthest away (Table S2).All plaque-to-plaque distances were significantly different from oneanother with the exception of the plakoglobin N-terminal and Dsg3C-terminal domains, and plakoglobin N-terminal and desmoplakinN-terminal domains (Fig. 2E). This is consistent with interactionsbetween plakoglobin and both the cytoplasmic tail of Dsg3 and theN-terminal domain of desmoplakin (Delva et al., 2009).Desmosome length, parallel to the plasma membrane, was also

determined for each protein domain (Fig. 2F). There was a trend ofincreasing lengthwith increasing distance from the plasmamembrane.This trend was also reflected at the protein domain level: thedesmoplakin C-terminal domain length was significantly longer thanthat of either the desmoplakin N-terminal or desmoplakin rod domainlength (Table S2). A composite molecular map including proteinlocalizations and desmosome length, illustrates the possiblearrangement of proteins splaying out from the membrane (Fig. 2G).We next sought to determine whether the arrangement of proteins

measured in primary human keratinocytes was conserved among theimmortalized human keratinocyte cell line HaCaT and the humanepidermoid carcinoma cell line A431. Individual desmoplakin rodand plakoglobin N-terminal-domain-labeled desmosomes wereimaged in all cell types and there were no significant differencesin plaque-to-plaque distances (Fig. S2). This suggests the generalorganization of proteins in desmosomes is conserved across primarykeratinocytes and human cell lines derived from skin.Desmosome protein organization has been previously studied

with electron microscopy, measured as distance from theplasma membrane. For comparison, dSTORM plaque-to-plaquemeasurements were converted into a plasma membrane referencesystem by subtracting the width of the intercellular space (∼34 nm);(Al-Amoudi et al., 2004), subtracting two times the plasmamembrane thickness (∼4-6 nm), and finally dividing by two (seeFig. 2G for reference). The dSTORM distributions of Dsg3 C-terminal and plakoglobin N-terminal domains overlap with thecorresponding distributions of gold labels in immunoelectron

microscopy studies (North et al., 1999), and the dSTORMplakoglobin distribution overlaps with the plakoglobin layeridentified by cryoelectron tomography (Al-Amoudi et al., 2011).In contrast to immunogold studies, dSTORM indicates thatdesmoplakin is localized further from the plasma membrane.

Although desmosome length has been previously measured withelectron microscopy, dSTORM allows for the analysis of specificprotein domains. The corresponding increase in plaque length andplaque-to-plaque distance indicates that the desmosome splays out asit extends from the plasma membrane into the cell. Desmoplakin hastwo isoforms, desmoplakin I and desmoplakin II, each with globularN- and C-terminal domains separated by a central rod domain. Theaverage N- to C-terminal length of desmoplakin I is 162 nm anddesmoplakin II is 63 nm (O’Keefe et al., 1989). The distance betweenthe desmoplakin N- and C-terminal domains measured perpendicularto the plasma membrane by dSTORM was 54±4 nm (mean±s.e.m;desmoplakin N-terminal n=36; C-terminal n=40), on par withprevious measurements (North et al., 1999), whereas the length ofthe desmoplakin C-terminal plaque extends 77±13 nm beyond thedesmoplakin N-terminal plaque. This suggests desmoplakin isoriented with its long axis at an angle in the plaque, notperpendicular to the plasma membrane. The minimum length thatwill fit this model is 94 nm, significantly less than the length ofdesmoplakin I. The desmoplakin II rod domain could span the 54 nm,but at a smaller angle. Although we cannot rule out an additionaldisorganized or kinked conformation of the rod, our data stronglysuggests that desmoplakin is oriented at an angle in the plaque.

Reorganization of plaque proteins in PKP-1-mediatedhyperadhesive desmosomesDesmosomes can exist in a weaker Ca2+-dependent state and astronger Ca2+-independent or hyperadhesive state (Garrod et al.,2005; Kimura et al., 2007). Plakophilin 1 (PKP-1) promotesdesmosome formation by recruiting and clustering desmosomalproteins, and overexpression of PKP-1 has been shown to inducehyperadhesion in cultured keratinocytes (Bornslaeger et al., 2001;Hatzfeld et al., 2000; Tucker et al., 2014). To test whether plaqueorganization changes with adhesive state, adenoviral PKP-1–Mycoverexpression was used to shift desmosomes to a hyperadhesivestate. With PKP-1–Myc overexpression an approximate eightfoldincrease in PKP-1 protein level was observed compared to emptyvector control (Fig. S3). Keratinocytes were labeled with antibodiesagainst the plakoglobin N-terminal, Dsg3 C-terminal, desmoplakinN-terminal, desmoplakin rod and desmoplakin C-terminal domains(Fig. 3A). Cells were also labeled with anti-Myc antibody, andPKP-1–Myc expression was confirmed by widefield colocalizationanalysis. The plaque-to-plaque distance was measured frommultiple individual desmosomes for each protein domain(Fig. 3B). A significant decrease in the plaque-to-plaque distancewas measured for the Dsg3 C-terminal, desmoplakin rod anddesmoplakin C-terminal domains. In contrast the distribution ofplakoglobin and the desmoplakin N-terminal domain did notsignificantly change (Fig. 3C; Table S3). An increase in desmosomelength was observed with PKP-1–Myc overexpression, similar to inprevious studies (Fig. 3D; Table S4) (Tucker et al., 2014).

Our data show that the plaque-to-plaque distance for some, but notall, protein domains decreases in PKP-1-induced hyperadhesion.Notably, the desmoplakin N-terminal domain plaque-to-plaquedistance does not change with PKP-1–Myc overexpressioncompared to control, whereas the plaque-to-plaque distance ofboth the desmoplakin rod and C-terminal domains decreased. TheN-terminal plakin domain of desmoplakin has an ‘L’ conformation

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consisting of two perpendicular lobes separated by a flexible elbow,with a flexible linker connecting theN-terminal to the rod domain (Al-Jassar et al., 2011; Grum et al., 1999). Our data supports previously

proposed models in which a conformational changewithin the plakindomain elbow or the flexible linker allows for motion of the rod andC-terminal domains within the plaque, whereas the N-terminal

Fig. 2. Molecularmap of the desmosome as revealed by dSTORM. (A) Approximate binding locations of antibodies used for dSTORM in desmoglein 3 (Dsg3),desmoplakin (DP) or plakoglobin (PG). (B) Representative images of individual desmosomes labeled with the indicated antibodies, from 2–4 preparations. Scalebar: 500 nm. (C) Fluorescence intensity linescan along the desmosomal axis (horizontal through image as shown). (D) Histograms of plaque-to-plaquedistance from all desmosomes analyzed for each protein domain. (E) Box-and-whisker plot of plaque-to-plaque distance. The box represents the 25–75thpercentiles, and the median is indicated. The whiskers show the 10–90th percentiles. (F) Desmosome length. Mean±s.e.m. *P<0.05 (one-way ANOVA withHolm-Sidak’s multiple comparisons test). For D–F, Dsg3 N-term, n=69; PG N-term, n=47; Dsg3 N-term, n=32; DP N-term, n=36; DP rod, n=28; DP C-term, n=40.(G) Composite dSTORM map where 0 is the midline, distances represent half the mean plaque-to-plaque distance, oval height represents desmosome length,and oval width represents the standard deviation of the peak width. PM, plasma membrane.

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domain is stationary (Al-Jassar et al., 2011). PKP-1 has also beenshown to interfere with plakoglobin-desmoplakin binding(Bornslaeger et al., 2001) and thus it is possible that the proposedconformational change also reflects a change in binding partner.The difference in ultrastructure measured with dSTORM could

contribute to the increased adhesive strength and mechanism ofdesmosome maturation. Interestingly, knockdown of PKP-3 inSCC9 cells has been shown to weaken cell adhesion and increasedesmosome width (Todorovic et al., 2014). That result, along withthose presented here, support the hypothesis that changes in proteinorganization within the desmosomal plaque correlate with changesin adhesive strength.

Desmosome molecular organization is conserved in skinTo investigate whether desmosomal plaques could be identifiedin vivo, human skin sections labeled with antibodies against thedesmoplakin rod domain were imaged by dSTORM. The epidermisis a stratified epithelium, and imaging the desmoplakin rod domainrevealed pairs of individual plaques in basal cells adjacent to thebasement membrane and suprabasal cells in the spinous andgranular layers (Fig. 4A).To quantify the protein arrangement in skin, the plakoglobin

N-terminal, desmoplakin rod or desmoplakin C-terminal domainswere labeled and imaged by dSTORM. The plaque-to-plaquedistances revealed that the overall axial arrangement of protein

domains was similar in skin and cell culture. Next, we wanted to seewhether the arrangement was the same in basal and suprabasal cells.Plaque-to-plaque distances measured in basal and suprabasaldesmosomes for plakoglobin N-terminal or desmoplakin rod werenot significantly different. However, for the desmoplakin C-terminaldomain, the plaque-to-plaque distance decreased significantly by65±11 nm (Fig. 4C; Table S5).

Expression levels of desmosomal proteins changewith keratinocytedifferentiation in the epidermis (Delva et al., 2009). PKP-1 expressionis increased in suprabasal compared to basal cells, and the decrease indesmoplakin C-terminal plaque-to-plaque distance in thesedesmosome agrees with our PKP-1–Myc overexpression results incell culture. However, the desmoplakin rod domain plaque-to-plaquedistance was unchanged between basal and suprabasal desmosomes,which could reflect variations in plaque re-organization due to proteinoverexpression verses epithelial differentiation.

In summary, our results demonstrate the ability of dSTORM toinvestigate the nanoscale arrangement of proteins within intactdesmosomal plaques, in cell culture and human tissue. Desmosomesare dynamic, exist in different adhesive states and can be disrupted bydisease. Therefore, this resolution will likely prove powerful inunderstanding the relationship between molecular organization anddesmosomal adhesive state (Garrod et al., 2005; Getsios et al., 2004;Green et al., 2010; Kitajima, 2014). The approach developed here isapplicable to many aspects of desmosome biology including their

Fig. 3. PKP-1-mediated hyperadhesion altered themolecularmap of the desmosome. (A) dSTORM images of individual desmosomes from cultured primarykeratinocytes transduced with control empty virus or PKP-1–Myc and labeled with indicated antibodies against plakoglobin (PG), Dsg3 or desmoplakin (DP).Images are representative of two experiments. Scale bar: 500 nm. (B) Histograms of the plaque-to-plaque distance measured in control (solid) and PKP-1–Myc(open). (C) Box-and-whisker plot of axial plaque-to-plaque distance of all protein domains analyzed in control (solid) and PKP-1–Myc overexpressing(striped) cells. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles (for B,C, control: PG N-term,n=14; Dsg3 C-term, n=33; DP N-term, n=50; DP rod, n=74; DP C-term, n=54; for PKP-1–Myc: PG N-term, n=19; Dsg3 C-term n=47; DP N-term n=20; DP rodn=61; DP C-term n=43). *P<0.05, **P<0.0001; n.s., not significant (paired t-tests). (D) Desmosome length (mean±s.e.m., control: PG N-term, n=81; DP rod,n=116. PKP-1–Myc: PG N-term, n=90; DP rod, n=95) measured for the plakoglobin N-terminal and desmoplakin rod domains in control (solid) and PKP-1–Mycoverexpressing (striped) cells. *P<0.05 (paired t-tests).

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interaction with intermediate filaments, the impact of intracellularsignaling on plaque organization and disruption in disease. Withrelatively straightforward sample preparation, compatibility with cellsand tissues, and single-desmosome resolution, this approach is poisedto integrate functional and structural analysis of desmosomes.

MATERIALS AND METHODSCell cultureHuman epidermal keratinocytes were isolated from neonatal foreskin aspreviously described (Calkins et al., 2006) and cultured in supplementedKBM-Gold (Lonza, Walkersville, MD). Keratinocytes, at passage two orthree, were seeded onto eight-well no. 1.5 coverslip bottommicroslides (IbidiGmbH, Planegg-Martinsried, Germany). The Ca2+ concentration in themediumwas increased to 550 µMat 16–20 h before fixation.Where indicated,keratinocytes were infected with empty or PKP-1–Myc adenovirus (Tuckeret al., 2014) 36 h before fixation. HaCaT and A431 cells were cultured inDulbecco’s modified Eagle’s medium (DMEM; Corning, Tewksbury, MA)supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

AntibodiesAntibodies used were: anti-Dsg3 AK15 (Tsunoda et al., 2003) (gift fromDr Masayuki Amagai, Keio University, Tokyo, Japan); anti-Dsg3 G194(Progen Biotechnik GmbH, Heidelberg); anti-γ-catenin (Santa CruzBiotechnology); anti-desmoplakin I and II (Fitzgerald, Acton, MA);desmoplakin antibodies NW6 and NW161 (gift from Dr Kathleen Green,Northwestern University, Evanston, IL) and anti-Myc (Bethyl Labs,Montgomery, TX). Additional information is in Table S1. Alexa-Fluor-conjugated secondary antibodies (IgG H&L) were from Invitrogen (GrandIsland, NY). For western blotting, anti-PKP-1 (Sobolik-Delmaire et al.,2006) (gift from Dr James Wahl III, University of Nebraska) and anti-ActinC2 (Santa Cruz Biotechnology) were used.

ImmunofluorescenceFixation and labeling protocols were adapted fromWhelan and Bell (2015).Cells were pre-extracted (60 s) with 300 mM sucrose and 0.2% Triton-X100 to remove non-desmosomal proteins. This was followed by fixation

(12 min, 37°C) with 4% paraformaldehyde prepared fresh from 16%electron microscopy grade material (Electron Microscopy Sciences,Hatfield, PA). Samples were washed, blocked and permeabilized (30 min)in 3% bovine serum albumin (BSA) and 0.2% Triton-X 100. Samples wereincubated with primary (3 h) and secondary antibodies (1 h), with multiplewashes in-between. Samples were stored with protection from light (PBS+,4°C) and imaged within 2 weeks.

Human tissue biopsy processingHuman skin biopsy use was approved by the Emory University InstitutionalReview Board. 5-µm-thick sections from OCT-embedded biopsies weremounted onto glass coverslips and processed for immunostaining. Tissuesections were labeled sequentially with primary and secondary antibodies(1 h) with triple PBS+ washes between incubations.

ImmunoblottingCells were harvested and immunoblotting performed using standardprocedures (Stahley et al., 2014).

MicroscopySIM imageswere obtained and reconstructed with a NikonN-SIMEclipse Ti-E microscope system (Nikon Instruments, Melville, NY) equipped with a100×1.49 NA oil immersion objective, 488 nm laser and EMCCD camera(DU-897, Andor Technology, Belfast, Northern Ireland). dSTORM imageswere obtainedwith aNikonN-STORMmicroscope equippedwith a 100×1.49NA oil immersion objective, 488 and 647 nm lasers, and an iXon ultraEMCCD camera (Andor). Typically, 60,000–80,000 frames were collectedwith inclined sub-critical excitation. Images were reconstructed in NikonElements. Widefield images were obtained with low-intensity laser excitationon the N-STORM system. Photoswitching imaging buffer included glucoseoxidase (Sigma, St Louis, Missouri), catalase (Roche, Penzberg, Germany),glucose (Sigma) and β-mercaptoethanol (Sigma) (Rust et al., 2006).

Image analysisdSTORM images were exported at 4 nm/pixel and analyzed usingMATLAB scripts (Mathworks, Natic, MA). Individual desmosomes were

Fig. 4. Molecular organization of desmosomes in human skin. (A) Human skin sections labeled for desmoplakin (DP, rod domain) with multiple desmosomesvisible throughout the epidermis in basal (bottom) and suprabasal (top) cells. Scale bar: 5 µm. (B) Representative images of individual desmosomes labeled withthe indicated antibodies from basal (bottom) and suprabasal (top) cells. PG, plakoglobin. Scale bar: 200 nm. (C) Box-and-whisker plot of plaque-to-plaquedistance in basal (solid) and suprabasal (striped) cells. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90thpercentiles (Basal: PG N-term, n=33; DP rod, n=80; DP C-term, n=29. Suprabasal: PG N-term, n=17; DP rod, n=34; DP C-term, n=12). *P<0.05 (paired t-tests).

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manually identified and automatically excised and aligned such that thedesmosome axis was horizontal. Intensity was measured along thedesmosome axis by averaging all pixels along the desmosome length.Linescans were normalized, smoothed and the peak finder function (40%intensity peak threshold) identified linescans with two peaks forquantification. For the Dsg3 N-terminal, linescans with one peak wereselected. Custom MATLAB scripts are available upon request.

AcknowledgementsWe thank Mattheyses and Kowalczyk laboratory members for discussion, SusanSummers for keratinocyte isolations and Sue Manos for aid with human tissue.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsS.N.S., E.I.B., A.P.K. and A.L.M. designed experiments; S.N.S., E.I.B. and A.L.M.performed the experiments, C.E.A. and A.L.M. wrote programs and analyzed datawith S.N.S. S.N.S. and A.L.M. wrote the manuscript. All authors contributedintellectually and edited the manuscript.

FundingThis project was supported by the National Institutes of Health [grant numbersR21AR066920 to A.L.M., R01AR048266 to A.P.K.]; andNational Science Foundation[grant number IDBR1353939 toA.L.M.]. SIMwas supported by the IntegratedCellularImaging Core at Emory University. Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.185785.supplemental

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