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. 2015 May 11;209(3):403-18.
doi: 10.1083/jcb.201502024.

Nuclear-cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions

Rachel M Stewart  1 , Amanda E Zubek  2 , Kathryn A Rosowski  1 , Sarah M Schreiner  1 , Valerie Horsley  3 , Megan C King  4
Affiliations

Nuclear-cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions

Rachel M Stewart et al. J Cell Biol. .

Abstract

The linker of nucleoskeleton and cytoskeleton (LINC) complex allows cells to actively control nuclear position by coupling the nucleus to the cytoplasmic cytoskeleton. Nuclear position responds to the formation of intercellular adhesions through coordination with the cytoskeleton, but it is not known whether this response impacts adhesion function. In this paper, we demonstrate that the LINC complex component SUN2 contributes to the mechanical integrity of intercellular adhesions between mammalian epidermal keratinocytes. Mice deficient for Sun2 exhibited irregular hair follicle intercellular adhesions, defective follicle structure, and alopecia. Primary mouse keratinocytes lacking Sun2 displayed aberrant nuclear position in response to adhesion formation, altered desmosome distribution, and mechanically defective adhesions. This dysfunction appeared rooted in a failure of Sun2-null cells to reorganize their microtubule network to support coordinated intercellular adhesion. Together, these results suggest that cross talk between the nucleus, cytoskeleton, and intercellular adhesions is important for epidermal tissue integrity.

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Figures

Figure 1.
Figure 1.
Sun2−/− mice exhibit alopecia and abnormal hair follicle structure. (A) Immunostaining of mouse back skin revealed NE localized SUN1 (also greyscale inset, arrows) was expressed in the epidermis. Asterisk indicates nonspecific staining. Dotted lines delineate hair follicles. (B) SUN2 was strongly expressed in the epidermis, dermis, and hair follicle. Dotted lines delineate hair follicles. Bar, 100 μm. (C) Sun2−/− mice developed alopecia at P16 yet regained a normal coat by the second hair cycle (P32). (D) Hematoxylin and eosin–stained back skin from WT and Sun2−/− mice show hair shaft breaks at P16 (IV and VI, arrow) and P18 (VIII and X, arrow). (E) Quantification of broken follicles in WT and Sun2−/− mice. n > 160 follicles in three mice per genotype. (F and G) Immunostaining of back skin from WT and Sun2−/− mice for keratin 6 (K6) and Hoechst illustrated acute bends in Sun2−/− follicles (G, arrows), whereas WT follicles remained linear (F, arrow). (H) Percentage of total follicles with at least one bend <130°. n > 98 follicles in three mice per genotype. Error bars indicate SDs. Statistical significance determined by unpaired, two-tailed t test.
Figure 2.
Figure 2.
Adhesion-dependent nuclear movement occurs in WT epidermal MKCs and is exaggerated in Sun2−/− MKCs. (A) SUN2 and E-cadherin (E-cad) localization in WT MKCs in low calcium (Ca2+) or in high Ca2+ medium for 24 h. (B) Diagram of a MKC colony illustrating interior adhesions (magenta) at cell–cell contacts opposite from the free edge in cells at the colony periphery. Nuclear position (asterisks) is biased toward interior adhesions and away from the cell centroid (marked with x’s). (C and D) E-cad and nuclear position in WT MKCs cultured in high Ca2+ medium for 0 and 24 h. Each cell periphery is outlined (dotted lines), and the nuclear centroid (asterisks) and cell centroid (x’s) are shown in matching colors. Arrow in D indicates a cell without a free edge with a central nuclear position. (E) Plots of the nuclear centroid to cell centroid distance normalized to cell radius during MKC adhesion formation at indicated time points after Ca2+ addition. n > 50 cells per time point (representative of three experiments). Error bars indicate SDs. Asterisks denote indicated significance (*, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ANOVA with Tukey’s posttest). (F and G) E-cad and nuclear position in WT and Sun2−/− MKCs cultured in high Ca2+ medium for 24 h. Nuclei (asterisks) directly abut E-cad–positive AJs (dotted lines) in Sun2−/− MKCs at colony edges (G, inset). (H) Quantification of nuclear position as in E for WT and Sun2−/− MKCs. n > 50 cells per genotype at each time point (representative of three experiments). Error bars indicate SDs. Statistical significance determined by unpaired, two-tailed t test. AU, arbitrary unit.
Figure 3.
Figure 3.
Adhesion-dependent nuclear movement is differentially regulated by the actin and MT cytoskeletons. (A) Diagram illustrating treatment of WT MKCs with the indicated drug before Ca2+ addition in B–D. (B) E-cadherin (E-cad; green), β-tubulin (magenta), actin (phalloidin, red), and nuclei (Hoechst) localization are shown. Diagrams illustrate the effect of drug treatment on nuclear position. (C and D) Plots of the nuclear centroid to cell centroid distance normalized to cell radius. n > 50 cells per condition (representative of three experiments). Nuclei in blebbistatin-treated cells remained located at the cell center. Nuclei in nocodazole-treated cells moved closer to adhesions than in DMSO-treated cells, which was rescued by blebbistatin treatment. (E) Diagram illustrating treatment of WT MKCs with the indicated drug after Ca2+ addition in F–H. (F) Staining as in B. (G and H) Quantification as in C and D. n > 50 cells per condition (representative of three experiments). Nuclear position was unaltered in blebbistatin-treated cells, whereas latrunculin A reduced nuclear movement. Nuclei in nocodazole-treated cells moved closer to adhesions than in control cells, which were partially rescued by blebbistatin. Asterisks denote indicated significance (**, P ≤ 0.01; ****, P ≤ 0.0001) as assessed by unpaired, two-tailed t test (C), ANOVA with Tukey’s posttest (D and H), or ANOVA with Dunnett’s posttest (G). All data plotted as box and whiskers plots; the bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the means. AU, arbitrary unit; Noco, nocodazole; Blebb, blebbistatin; Lat A, Latrunculin A.
Figure 4.
Figure 4.
Organization of the MT cytoskeleton is perturbed in differentiated Sun2−/− keratinocytes. (A) Localization of β-tubulin, phosphorylated myosin light chain (p-MLC), keratin 14 (K14), actin (phalloidin), and nuclei (Hoechst) in WT MKCs. (B) Insets of β-tubulin localization in white boxes in A and D. Arrowheads indicate MT organization at the cell periphery. (C) Quantification of the percentage of WT or Sun2−/− MKCs in the indicated media with cortical accumulation of MTs. n > 200 cells per condition or genotype from three experiments. Asterisks denote indicated significance (**, P < 0.01; ANOVA with Dunnett’s posttest). Error bars indicate SDs. (D) Staining as in A for Sun2−/− MKCs in high Ca2+ medium after 24 h. Arrows in β-tubulin images indicate a perinuclear β-tubulin cage in WT cells that is often lacking in Sun2−/− MKCs. Arrows in K14 images indicate a perinuclear K14 cage in both WT and Sun2−/− MKCs. (E) Localization of β-tubulin, E-cad, and nuclei (Hoechst) after 5 h of taxol treatment in WT or Sun2−/− MKCs. Arrows indicate intercellular junctions (β-tubulin images), cell periphery (I and II), or NE (III and IV). Note the lack of MT accumulation at these sites in Sun2−/− MKCs. Dashed lines indicate the colony periphery defined by E-cad staining. (F) Quantification of the percentage of WT and Sun2−/− MKCs with a stabilized pool of NE-associated MTs. n > 200 cells from three experiments. Error bars indicate SDs. Statistical significance determined by unpaired, two-tailed t test. (G) Actin (phalloidin), E-cad, and nuclei (Hoechst) localization in Sun2−/− MKCs in high Ca2+ medium for 24 h treated with the indicated drug for 5 h. Diagrams illustrate the effect of drug treatment on nuclear position. (H) Plots of nuclear centroid to cell centroid distance normalized to cell radius. Latrunculin A partially rescued the excessive nuclear movement of Sun2−/− MKCs. n > 50 cells (representative of three experiments). Asterisks denote indicated significance (**, P = 0.0087; ANOVA with Dunnett’s posttest). The bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the mean. AU, arbitrary unit; cal, calcium; Blebb, blebbistatin; Lat A, Latrunculin A.
Figure 5.
Figure 5.
Cultured Sun2−/− keratinocytes form altered, mechanically weak intercellular adhesions. (A and B) WT or Sun2−/− MKCs in high Ca2+ medium after 72 h revealed desmosome (DPI/II) and AJ (E-cad) formation. (B) Insets from A show adhesion formation in WT and Sun2−/− MKCs (arrowheads). (C) Western blot analysis of Triton X-100–soluble and –insoluble fractions of WT and Sun2−/− MKC cell lysates, cultured in low or high Ca2+ medium, and probed for desmoglein 3 (Dsg3), desmoplakin I/II (DPI/II), and β-actin (loading control). (D) DPI/II (red), E-cad (green), and nuclear (Hoechst) localization in WT and Sun2−/− MKCs after 24 h in high Ca2+ medium revealed a uniform distribution of DPI/II along interior and lateral junctions in WT cells. This pattern of localization was perturbed in Sun2−/− cells. Insets display single lateral adhesions indicated by white boxes. (E) Diagram illustrating the location of interior (magenta) and lateral (blue) cell–cell adhesions in MKCs at the edge of colonies. Black arrow indicates direction of measurements in H. (F) The ratio of interior junction DPI/II intensity per micrometer to lateral junction DPI/II intensity per micrometer in WT and Sun2−/− MKCs after 24 h in high Ca2+ medium or for WT cells pretreated with DMSO or nocodazole before Ca2+ addition. Both Sun2−/− and nocodazole-treated MKCs displayed a significantly increased distribution of DPI/II at interior junctions. n > 67 cells per genotype and condition from three experiments. Statistical significance determined by unpaired, two-tailed t test. The bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the mean. (G) Averaged line scans of DPI/II intensity (arbitrary units [AU]) measured orthogonally across WT and Sun2−/− interior junctions for images as in D. n > 26 cells per genotype, three measurements per adhesion. (H) Line scans of DPI/II intensity (arbitrary units) along single representative WT and Sun2−/− MKC lateral adhesions, extending from the interior junctions to the cell edge (representative of n > 106 junctions from three experiments). (I and J) WT monolayers remained intact (I), whereas Sun2−/− monolayers fragmented drastically (J) after 72 h in high Ca2+ medium followed by mechanical challenge. (K) Quantification of the adhesion integrity assay in I and J. Error bars indicate SD for two replicates of three monolayers per genotype. Statistical significance determined by unpaired, two-tailed t test. AU, arbitrary unit; Noco, nocodazole.
Figure 6.
Figure 6.
Intercellular adhesions are structurally and functionally perturbed in Sun2−/− hair follicles. Transmission electron micrographs of P4 WT and Sun2−/− follicles. (A and B) Overview of sectioned follicle structure, with the ORS at the edges of the image, followed by the companion (Cp), Henle (He), and Huxley (Hu) layers. Note the gaps between Sun2−/− He cells (B, arrows). (C and D) WT He–He cell junctions were evenly spaced with classical electron-dense desmosome morphology (C and C′, arrow), whereas Sun2−/− junctions included large gaps between cells, abnormal desmosome structure, and fewer desmosomes (D and D′, arrow). (E) Quantification of intermembrane spacings between He cells, displayed on a log2 scale. n > 138 adhesions from two mice. (F) Quantification of linear desmosome density between He layer cells. n > 13 cells from two mice. (G) Quantification of desmosome length between He layer cells. n > 62 desmosomes from two mice. (H and I) A greater number of desmosomes (insets H′ and I′, arrows) were found between He and Hu layer cells in Sun2−/− follicles. (J) Quantification of linear desmosome density between He and Hu cells. n > 19 cells from two mice. (K and L) Back skin from P4 WT and Sun2−/− mice immunostained for Dsg4. Junctions between He and Hu layers were desmosome rich (boxes). (M) Mean fluorescence intensity per micrometer for stretches of He–Hu junctions. n > 13 junctions. In all cases, statistical significance was determined by unpaired, two-tailed t test. For box and whiskers plots, the bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the means.
Figure 7.
Figure 7.
Model of the effect of Sun2 loss on intercellular adhesion and cytoskeletal organization in vitro and in vivo. (A) Diagram depicting the organization of cytoskeletal elements, forces on nuclei (F in arrows), and desmosome distribution in differentiated WT and Sun2−/− MKCs. In WT cells, the MT and actin cytoskeletons appear to impose opposing forces on the nucleus, resulting in a bias in nuclear position toward interior adhesions. Sun2−/− cells display defective MT and actin organization; alterations in MT dynamics in Sun2−/− cells most likely produce the observed defect in nuclear positioning. An even distribution of desmosomes along interior and lateral intercellular adhesions is disrupted in Sun2−/− cells. (B) Sun2−/− hair follicles exhibit a desmosome deficiency along lateral-like He–He junctions, along with alterations in desmosome morphology and intermembrane spacing, as well as an increase in desmosomes along interior-like He–Hu junctions compared with WT follicles. These changes in desmosome density parallel the changes in desmosome distribution observed in vitro.
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