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Home , Desmosome, Focal adhesion, Hemidesmosome

© 2016. Published by The Company of Biologists Ltd | Journal of Science (2016) 129, 2897-2904 doi:10.1242/jcs.185785

SHORT REPORT Molecular organization of the as revealed by direct stochastic optical reconstruction microscopy Sara N. Stahley1, Emily I. Bartle1, Claire E. Atkinson2, Andrew P. Kowalczyk1,3 and Alexa L. Mattheyses1,*

ABSTRACT and , and the family member are macromolecular junctions responsible for providing contribute to the intracellular plaque (Fig. 1A). strong cell–. Because of their size and molecular Plaque ultrastructure is characterized by two electron-dense complexity, the precise ultrastructural organization of desmosomes regions: the plasma-membrane-proximal outer dense plaque and is challenging to study. Here, we used direct stochastic optical the inner dense plaque (Desai et al., 2009; Farquhar and Palade, reconstruction microscopy (dSTORM) to resolve individual plaque 1963; Stokes, 2007). The cytoplasmic tails bind to proteins pairs for inner and outer dense plaque proteins. Analysis methods in the outer dense plaque whereas the C-terminus of desmoplakin based on desmosomal mirror symmetry were developed to measure binds to intermediate filaments in the inner dense plaque. This plaque-to-plaque distances and create an integrated map. We tethers the desmosome to the , quantified the organization of 3, plakoglobin and establishing an integrated adhesive network (Bornslaeger et al., desmoplakin (N-terminal, rod and C-terminal domains) in primary 1996; Harmon and Green, 2013). human . Longer desmosome lengths correlated with Many desmosomal protein interactions have been characterized increasing plaque-to-plaque distance, suggesting that desmoplakin is by biochemical studies (Bass-Zubek and Green, 2007; Green and arranged with its long axis at an angle within the plaque. We Simpson, 2007; Thomason et al., 2010), and desmosomal next examined whether plaque organization changed in different ultrastructure has been studied by immunogold and tomographic adhesive states. Plaque-to-plaque distance for the desmoplakin electron microscopy (Al-Amoudi et al., 2011; Al-Amoudi and rod and C-terminal domains decreased in PKP-1-mediated Frangakis, 2008; North et al., 1999; Owen et al., 2008; Stokes, hyperadhesive desmosomes, suggesting that protein reorganization 2007). Yet, studying desmosomes on a molecular level remains correlates with function. Finally, in human we found a challenging due to their size, insolubility and molecular complexity. difference in plaque-to-plaque distance for the desmoplakin C- Measuring desmosomal protein organization requires a technique terminal domain, but not the desmoplakin rod domain or with high resolution coupled with minimal sample manipulation to plakoglobin, between basal and suprabasal cells. Our data reveal minimize structural artifacts and preserve physiological relevance. the molecular organization of desmosomes in cultured keratinocytes Super-resolution microscopy has revealed sub-resolution protein and as defined by dSTORM. organization in numerous macromolecular complexes including the nuclear pore, ciliary transition zone, kinetochore, neuronal synapse, KEY WORDS: Cell adhesion, , Super-resolution, and (Chatel et al., 2012; Fluorescence, Microscopy, Cadherin, dSTORM Loschberger et al., 2014, 2012; Nahidiazar et al., 2015; Ribeiro et al., 2010; van Hoorn et al., 2014; Yang et al., 2015; Zhong, 2015). INTRODUCTION Here, we use direct stochastic optical reconstruction microscopy Desmosomes are intercellular junctions that are abundant in tissues (dSTORM) (Rust et al., 2006) to elucidate protein organization that experience considerable mechanical stress, such as the skin within the desmosome at the molecular level both in vitro and and heart (Kowalczyk and Green, 2013). The importance of in vivo. desmosome-mediated adhesion is evidenced by the numerous human diseases that result when desmosomes are compromised, RESULTS AND DISCUSSION manifesting in epidermal fragility, cardiomyopathies and cancers Desmosome plaques resolved by dSTORM (Broussard et al., 2015; Stahley and Kowalczyk, 2015). Two proteins, the inner dense plaque protein desmoplakin and the The desmosome is a macromolecular complex that forms a ‘spot- outer dense plaque protein plakoglobin, were utilized to establish weld’ of strong intercellular adhesion and comprises transmembrane dSTORM for studying desmosome organization. Primary human and cytoplasmic plaque proteins (Berika and Garrod, keratinocytes were pre-extracted prior to fixation and the 2014; Saito et al., 2012). The desmosomal cadherins ( desmosomal pool of proteins (Palka and Green, 1997) was and ) link neighboring cells through extracellular immunostained with primary antibodies against the desmoplakin adhesive interactions whereas the armadillo protein family members C-terminal or plakoglobin N-terminal domains. The same cell–cell border was then imaged by widefield, structured illumination 1Department of , Emory University School of Medicine, Atlanta, GA microscopy (SIM) and dSTORM. 30322, USA. 2Department of Biochemistry and Molecular Biology, University of In widefield, desmoplakin fluorescence was punctate and Chicago, Chicago, IL 60637, USA. 3Department of Dermatology, Emory University School of Medicine, Atlanta, GA 30322, USA. pixilated with no discernable structural features. In contrast, two distinct desmoplakin plaques, one contributed by each cell, were *Author for correspondence ([email protected]) resolved by SIM as previously shown (Fig. 1B,C) (Stahley et al., A.L.M., 0000-0002-5119-7750 2015). Likewise, two individual desmoplakin plaques were resolved by dSTORM, but with a more distinct separation (Fig. 1C).

Received 7 January 2016; Accepted 15 June 2016 To analyze the difference in spatial resolution between the imaging Journal of Cell Science

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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 cell border. (C) Desmoplakin-labeled region of interest imaged by widefield microscopy, SIM and dSTORM. (D) Linescan across a single desmosome indicated by the colored 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). techniques, fluorescence intensity was plotted as a function of plakoglobin plaques (Fig. 1F). This is also shown in the plot of distance along the desmosome axis, perpendicular to the fluorescence intensity along the desmosome axis (Fig. 1G). plasma membrane. Although there is only a single intensity peak To confirm dSTORM resolves individual desmosomes consisting from the widefield image, two individual peaks, corresponding to of plaques contributed by two different cells, keratinocytes were the two plaques, are resolved from the SIM and dSTORM images dual-labeled for the extracellular domain of desmoglein 3 (Dsg3) (Fig. 1D). and the C-terminal domain of desmoplakin. dSTORM revealed a Plakoglobin-labeled desmosomes imaged by widefield have a distinct separation, with the Dsg3 extracellular domain sandwiched punctate appearance, similar to desmoplakin (Fig. 1E,F). Similarly, between two desmoplakin plaques (Fig. 1H). In our experiments, plakoglobin fluorescence by SIM is punctate with no evident the localization precision of Alexa Fluor 488 (∼19.5 nm) was less internal structure. In contrast, dSTORM revealed two individual than that of Alexa Fluor 647 (∼5.8 nm), similar to that reported by Journal of Cell Science

2898 SHORT REPORT Journal of Cell Science (2016) 129, 2897-2904 doi:10.1242/jcs.185785 others (Dempsey et al., 2011; Tam et al., 2014). Therefore, Alexa microscopy studies (North et al., 1999), and the dSTORM Fluor 647 was used for quantitative measurements unless otherwise plakoglobin distribution overlaps with the plakoglobin layer indicated. Image resolution is also affected by the primary and identified by cryoelectron tomography (Al-Amoudi et al., 2011). secondary antibody labels, which increases the apparent size of In contrast to immunogold studies, dSTORM indicates that structures (Bates et al., 2007). desmoplakin is localized further from the plasma membrane. In summary, these results demonstrate that dSTORM can resolve Although desmosome length has been previously measured with proteins in both the outer and inner dense plaques in individual electron microscopy, dSTORM allows for the analysis of specific desmosomes. protein domains. The corresponding increase in plaque length and plaque-to-plaque distance indicates that the desmosome splays out as Molecular map of the desmosome by dSTORM it extends from the plasma membrane into the cell. Desmoplakin has Next, we systematically quantified the molecular arrangement of two isoforms, desmoplakin I and desmoplakin II, each with globular proteins in the desmosome. Keratinocytes were labeled with N- and C-terminal domains separated by a central rod domain. The antibodies against the Dsg3 N-terminal (Alexa Fluor 488), average N- to C-terminal length of desmoplakin I is 162 nm and plakoglobin N-terminal, Dsg3 C-terminal, desmoplakin N- desmoplakin II is 63 nm (O’Keefe et al., 1989). The distance between terminal, desmoplakin rod, and desmoplakin C-terminal domains the desmoplakin N- and C-terminal domains measured perpendicular (Fig. 2A; Table S1) and protein distributions along the desmosomal to the plasma membrane by dSTORM was 54±4 nm (mean±s.e.m; axis were determined using dSTORM. The mirror symmetry of desmoplakin N-terminal n=36; C-terminal n=40), on par with desmosomal plaques allowed us to identify individual desmosomes, previous measurements (North et al., 1999), whereas the length of and only those with their axes in the x-y focal plane were analyzed the desmoplakin C-terminal plaque extends 77±13 nm beyond the (Fig. 2B; Fig. S1). A linescan of fluorescence intensity along desmoplakin N-terminal plaque. This suggests desmoplakin is the desmosome axis was created for each desmosome (Fig. 2C). The oriented with its long axis at an angle in the plaque, not axial distribution of proteins was automatically measured as the perpendicular to the plasma membrane. The minimum length that distance between peaks from the linescans. This plaque-to-plaque will fit this model is 94 nm, significantly less than the length of distance was measured from multiple individual desmosomes desmoplakin I. The desmoplakin II rod domain could span the 54 nm, labeled for each protein domain (Fig. 2D). There was a but at a smaller angle. Although we cannot rule out an additional progressive increase in the average plaque-to-plaque distance with disorganized or kinked conformation of the rod, our data strongly the plakoglobin N-terminal domain closest to the plasma membrane suggests that desmoplakin is oriented at an angle in the plaque. and the desmoplakin C-terminal domain furthest away (Table S2). All plaque-to-plaque distances were significantly different from one Reorganization of plaque proteins in PKP-1-mediated another with the exception of the plakoglobin N-terminal and Dsg3 hyperadhesive desmosomes C-terminal domains, and plakoglobin N-terminal and desmoplakin Desmosomes can exist in a weaker Ca2+-dependent state and a N-terminal domains (Fig. 2E). This is consistent with interactions stronger Ca2+-independent or hyperadhesive state (Garrod et al., between plakoglobin and both the cytoplasmic tail of Dsg3 and the 2005; Kimura et al., 2007). Plakophilin 1 (PKP-1) promotes N-terminal domain of desmoplakin (Delva et al., 2009). desmosome formation by recruiting and clustering desmosomal Desmosome length, parallel to the plasma membrane, was also proteins, and overexpression of PKP-1 has been shown to induce determined for each protein domain (Fig. 2F). There was a trend of hyperadhesion in cultured keratinocytes (Bornslaeger et al., 2001; increasing length with increasing distance from the plasma membrane. Hatzfeld et al., 2000; Tucker et al., 2014). To test whether plaque This trend was also reflected at the protein domain level: the organization changes with adhesive state, adenoviral PKP-1–Myc desmoplakin C-terminal domain length was significantly longer than overexpression was used to shift desmosomes to a hyperadhesive that of either the desmoplakin N-terminal or desmoplakin rod domain state. With PKP-1–Myc overexpression an approximate eightfold length (Table S2). A composite molecular map including protein increase in PKP-1 protein level was observed compared to empty localizations and desmosome length, illustrates the possible vector control (Fig. S3). Keratinocytes were labeled with antibodies arrangement of proteins splaying out from the membrane (Fig. 2G). against the plakoglobin N-terminal, Dsg3 C-terminal, desmoplakin We next sought to determine whether the arrangement of proteins N-terminal, desmoplakin rod and desmoplakin C-terminal domains measured in primary human keratinocytes was conserved among the (Fig. 3A). Cells were also labeled with anti-Myc antibody, and immortalized human keratinocyte cell line HaCaT and the human PKP-1–Myc expression was confirmed by widefield colocalization epidermoid cell line A431. Individual desmoplakin rod analysis. The plaque-to-plaque distance was measured from and plakoglobin N-terminal-domain-labeled desmosomes were multiple individual desmosomes for each protein domain imaged in all cell types and there were no significant differences (Fig. 3B). A significant decrease in the plaque-to-plaque distance in plaque-to-plaque distances (Fig. S2). This suggests the general was measured for the Dsg3 C-terminal, desmoplakin rod and organization of proteins in desmosomes is conserved across primary desmoplakin C-terminal domains. In contrast the distribution of keratinocytes and human cell lines derived from skin. plakoglobin and the desmoplakin N-terminal domain did not Desmosome protein organization has been previously studied significantly change (Fig. 3C; Table S3). An increase in desmosome with electron microscopy, measured as distance from the length was observed with PKP-1–Myc overexpression, similar to in plasma membrane. For comparison, dSTORM plaque-to-plaque previous studies (Fig. 3D; Table S4) (Tucker et al., 2014). measurements were converted into a plasma membrane reference Our data show that the plaque-to-plaque distance for some, but not system by subtracting the width of the intercellular space (∼34 nm); all, protein domains decreases in PKP-1-induced hyperadhesion. (Al-Amoudi et al., 2004), subtracting two times the plasma Notably, the desmoplakin N-terminal domain plaque-to-plaque membrane thickness (∼4-6 nm), and finally dividing by two (see distance does not change with PKP-1–Myc overexpression Fig. 2G for reference). The dSTORM distributions of Dsg3 C- compared to control, whereas the plaque-to-plaque distance of terminal and plakoglobin N-terminal domains overlap with the both the desmoplakin rod and C-terminal domains decreased. The corresponding distributions of gold labels in immunoelectron N-terminal plakin domain of desmoplakin has an ‘L’ conformation Journal of Cell Science

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Fig. 2. Molecular map 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. Scale bar: 500 nm. (C) Fluorescence intensity linescan along the desmosomal axis (horizontal through image as shown). (D) Histograms of plaque-to-plaque distance from all desmosomes analyzed for each protein domain. (E) Box-and-whisker plot of plaque-to-plaque distance. The box represents the 25–75th percentiles, 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 with Holm-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. consisting of two perpendicular lobes separated by a flexible elbow, proposed models in which a conformational change within the plakin with a flexible linkerconnecting the N-terminal to the rod domain (Al- domain elbow or the flexible linker allows for motion of the rod and

Jassar et al., 2011; Grum et al., 1999). Our data supports previously C-terminal domains within the plaque, whereas the N-terminal Journal of Cell Science

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Fig. 3. PKP-1-mediated hyperadhesion altered the molecular map of the desmosome. (A) dSTORM images of individual desmosomes from cultured primary keratinocytes 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 rod n=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–Myc overexpressing (striped) cells. *P<0.05 (paired t-tests). domain is stationary (Al-Jassar et al., 2011). PKP-1 has also been domains was similar in skin and cell culture. Next, we wanted to see shown to interfere with plakoglobin-desmoplakin binding whether the arrangement was the same in basal and suprabasal cells. (Bornslaeger et al., 2001) and thus it is possible that the proposed Plaque-to-plaque distances measured in basal and suprabasal conformational change also reflects a change in binding partner. desmosomes for plakoglobin N-terminal or desmoplakin rod were The difference in ultrastructure measured with dSTORM could not significantly different. However, for the desmoplakin C-terminal contribute to the increased adhesive strength and mechanism of domain, the plaque-to-plaque distance decreased significantly by desmosome maturation. Interestingly, knockdown of PKP-3 in 65±11 nm (Fig. 4C; Table S5). SCC9 cells has been shown to weaken cell adhesion and increase Expression levels of desmosomal proteins change with keratinocyte desmosome width (Todorovic et al., 2014). That result, along with differentiation in the epidermis (Delva et al., 2009). PKP-1 expression those presented here, support the hypothesis that changes in protein is increased in suprabasal compared to basal cells, and the decrease in organization within the desmosomal plaque correlate with changes desmoplakin C-terminal plaque-to-plaque distance in these in adhesive strength. desmosome agrees with our PKP-1–Myc overexpression results in cell culture. However, the desmoplakin rod domain plaque-to-plaque Desmosome molecular organization is conserved in skin distance was unchanged between basal and suprabasal desmosomes, To investigate whether desmosomal plaques could be identified which could reflect variations in plaque re-organization due to protein in vivo, human skin sections labeled with antibodies against the overexpression verses epithelial differentiation. desmoplakin rod domain were imaged by dSTORM. The epidermis In summary, our results demonstrate the ability of dSTORM to is a stratified , and imaging the desmoplakin rod domain investigate the nanoscale arrangement of proteins within intact revealed pairs of individual plaques in basal cells adjacent to the desmosomal plaques, in cell culture and human . Desmosomes and suprabasal cells in the spinous and are dynamic, exist in different adhesive states and can be disrupted by granular layers (Fig. 4A). disease. Therefore, this resolution will likely prove powerful in To quantify the protein arrangement in skin, the plakoglobin understanding the relationship between molecular organization and N-terminal, desmoplakin rod or desmoplakin C-terminal domains desmosomal adhesive state (Garrod et al., 2005; Getsios et al., 2004; were labeled and imaged by dSTORM. The plaque-to-plaque Green et al., 2010; Kitajima, 2014). The approach developed here is distances revealed that the overall axial arrangement of protein applicable to many aspects of desmosome biology including their Journal of Cell Science

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Fig. 4. Molecular organization of desmosomes in human skin. (A) Human skin sections labeled for desmoplakin (DP, rod domain) with multiple desmosomes visible throughout the epidermis in basal (bottom) and suprabasal (top) cells. Scale bar: 5 µm. (B) Representative images of individual desmosomes labeled with the indicated antibodies from basal (bottom) and suprabasal (top) cells. PG, plakoglobin. Scale bar: 200 nm. (C) Box-and-whisker plot of plaque-to-plaque distance in basal (solid) and suprabasal (striped) cells. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles (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). interaction with intermediate filaments, the impact of intracellular (12 min, 37°C) with 4% paraformaldehyde prepared fresh from 16% signaling on plaque organization and disruption in disease. With electron microscopy grade material (Electron Microscopy Sciences, relatively straightforward sample preparation, compatibility with cells Hatfield, PA). Samples were washed, blocked and permeabilized (30 min) and tissues, and single-desmosome resolution, this approach is poised in 3% bovine serum albumin (BSA) and 0.2% Triton-X 100. Samples were to integrate functional and structural analysis of desmosomes. incubated with primary (3 h) and secondary antibodies (1 h), with multiple washes in-between. Samples were stored with protection from light (PBS+, 4°C) and imaged within 2 weeks. MATERIALS AND METHODS Cell culture Human epidermal keratinocytes were isolated from neonatal foreskin as Human tissue biopsy processing Human skin biopsy use was approved by the Emory University Institutional previously described (Calkins et al., 2006) and cultured in supplemented Review Board. 5-µm-thick sections from OCT-embedded biopsies were KBM-Gold (Lonza, Walkersville, MD). Keratinocytes, at passage two or mounted onto glass coverslips and processed for immunostaining. Tissue three, were seeded onto eight-well no. 1.5 coverslip bottom microslides (Ibidi sections were labeled sequentially with primary and secondary antibodies GmbH, Planegg-Martinsried, Germany). The Ca2+ concentration in the (1 h) with triple PBS+ washes between incubations. medium was increased to 550 µM at 16–20 h before fixation. Where indicated, keratinocytes were infected with empty or PKP-1–Myc adenovirus (Tucker et al., 2014) 36 h before fixation. HaCaT and A431 cells were cultured in Immunoblotting Dulbecco’s modified Eagle’s medium (DMEM; Corning, Tewksbury, MA) Cells were harvested and immunoblotting performed using standard supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. procedures (Stahley et al., 2014).

Antibodies Microscopy Antibodies used were: anti-Dsg3 AK15 (Tsunoda et al., 2003) (gift from SIM images were obtained and reconstructed with a Nikon N-SIM Eclipse Ti- Dr Masayuki Amagai, Keio University, Tokyo, Japan); anti-Dsg3 G194 E microscope system (Nikon Instruments, Melville, NY) equipped with a (Progen Biotechnik GmbH, Heidelberg); anti-γ-catenin (Santa Cruz 100×1.49 NA oil immersion objective, 488 nm laser and EMCCD camera Biotechnology); anti-desmoplakin I and II (Fitzgerald, Acton, MA); (DU-897, Andor Technology, Belfast, Northern Ireland). dSTORM images desmoplakin antibodies NW6 and NW161 (gift from Dr Kathleen Green, were obtained with a Nikon N-STORM microscope equipped with a 100×1.49 Northwestern University, Evanston, IL) and anti-Myc (Bethyl Labs, NA oil immersion objective, 488 and 647 nm lasers, and an iXon ultra Montgomery, TX). Additional information is in Table S1. Alexa-Fluor- EMCCD camera (Andor). Typically, 60,000–80,000 frames were collected conjugated secondary antibodies (IgG H&L) were from Invitrogen (Grand with inclined sub-critical excitation. Images were reconstructed in Nikon Island, NY). For western blotting, anti-PKP-1 (Sobolik-Delmaire et al., Elements. Widefield images were obtained with low-intensity laser excitation 2006) (gift from Dr James Wahl III, University of Nebraska) and anti- on the N-STORM system. Photoswitching imaging buffer included glucose C2 (Santa Cruz Biotechnology) were used. oxidase (Sigma, St Louis, Missouri), catalase (Roche, Penzberg, Germany), glucose (Sigma) and β-mercaptoethanol (Sigma) (Rust et al., 2006). Immunofluorescence Fixation and labeling protocols were adapted from Whelan and Bell (2015). Image analysis Cells were pre-extracted (60 s) with 300 mM sucrose and 0.2% Triton-X dSTORM images were exported at 4 nm/pixel and analyzed using

100 to remove non-desmosomal proteins. This was followed by fixation MATLAB scripts (Mathworks, Natic, MA). Individual desmosomes were Journal of Cell Science

2902 SHORT REPORT Journal of Cell Science (2016) 129, 2897-2904 doi:10.1242/jcs.185785 manually identified and automatically excised and aligned such that the Garrod, D. R., Berika, M. Y., Bardsley, W. F., Holmes, D. and Tabernero, L. desmosome axis was horizontal. Intensity was measured along the (2005). Hyper-adhesion in desmosomes: its regulation in desmosome axis by averaging all pixels along the desmosome length. and possible relationship to cadherin crystal structure. J. Cell Sci. 118, 5743-5754. Getsios, S., Huen, A. C. and Green, K. J. (2004). Working out the strength and Linescans were normalized, smoothed and the peak finder function (40% flexibility of desmosomes. Nat. Rev. Mol. Cell Biol. 5, 271-281. intensity peak threshold) identified linescans with two peaks for Green, K. J. and Simpson, C. L. (2007). Desmosomes: new perspectives on a quantification. For the Dsg3 N-terminal, linescans with one peak were classic. J. Invest. Dermatol. 127, 2499-2515. selected. Custom MATLAB scripts are available upon request. Green, K. J., Getsios, S., Troyanovsky, S. and Godsel, L. M. (2010). Intercellular junction assembly, dynamics, and homeostasis. Cold Spring Harb. Perspect. Biol. Acknowledgements 2, a000125. ́ We thank Mattheyses and Kowalczyk laboratory members for discussion, Susan Grum, V. L., Li, D., MacDonald, R. I. and Mondragon, A. (1999). Structures of two repeats of spectrin suggest models of flexibility. Cell 98, 523-535. Summers for keratinocyte isolations and Sue Manos for aid with human tissue. Harmon, R. M. and Green, K. J. (2013). Structural and functional diversity of desmosomes. Cell Commun. Adhes. 20, 171-187. Competing interests Hatzfeld, M., Haffner, C., Schulze, K. and Vinzens, U. (2000). The function of The authors declare no competing or financial interests. plakophilin 1 in desmosome assembly and actin filament organization. J. Cell Biol. 149, 209-222. Author contributions Kimura, T. E., Merritt, A. J. and Garrod, D. R. (2007). Calcium-independent S.N.S., E.I.B., A.P.K. and A.L.M. designed experiments; S.N.S., E.I.B. and A.L.M. desmosomes of keratinocytes are hyper-adhesive. J. Invest. Dermatol. 127, 775-781. th performed the experiments, C.E.A. and A.L.M. wrote programs and analyzed data Kitajima, Y. (2014). 150 anniversary series: desmosomes and autoimmune with S.N.S. S.N.S. and A.L.M. wrote the manuscript. All authors contributed disease, perspective of dynamic desmosome remodeling and its impairments in intellectually and edited the manuscript. . Cell Commun. Adhes. 21, 269-280. Kowalczyk, A. P. and Green, K. J. (2013). Structure, function, and regulation of Funding desmosomes. Prog. Mol. Biol. Transl. Sci. 116, 95-118. Loschberger, A., van de Linde, S., Dabauvalle, M.-C., Rieger, B., Heilemann, M., This project was supported by the National Institutes of Health [grant numbers Krohne, G. and Sauer, M. (2012). Super-resolution imaging visualizes the eightfold R21AR066920 to A.L.M., R01AR048266 to A.P.K.]; and National Science Foundation symmetry of gp210 proteins around the nuclear pore complex and resolves the [grant number IDBR 1353939 to A.L.M.]. SIM was supported by the Integrated Cellular central channel with nanometer resolution. J. cell Sci. 125, 570-575. Imaging Core at Emory University. Deposited in PMC for release after 12 months. Loschberger, A., Franke, C., Krohne, G., van de Linde, S. and Sauer, M. (2014). Correlative super-resolution fluorescence and electron microscopy of the nuclear Supplementary information pore complex with molecular resolution. J. cell Sci. 127, 4351-4355. Supplementary information available online at Nahidiazar, L., Kreft, M., van den Broek, B., Secades, P., Manders, E. M. M., http://jcs.biologists.org/lookup/doi/10.1242/jcs.185785.supplemental Sonnenberg, A. and Jalink, K. (2015). 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