Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Abstract

Desmosomes are cell-cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.

Figure 1.

A subset of centrosomal proteins is recruited to the desmosome. (A–L) Localization of Ndel1 and Lis1 in mouse keratinocytes. (A–C) Anti-Ndel1 (red) and γ-tubulin (green) immunofluorescence staining in WT mouse keratinocytes cultured in low Ca²⁺. (D–F) Anti-Lis1 (red) and γ-tubulin (green) staining in WT mouse keratinocytes cultured in low Ca²⁺. A–F are maximum intensity projections. (G) Anti-Ndel1 (green) and anti-DP (red) staining in WT mouse keratinocytes. The boxed region is enlarged on the right to demonstrate the colocalization of Ndel1 and DP. (H) Anti-Lis1 (red) and anti-DP (green) staining in WT mouse keratinocytes. The boxed region is enlarged on the right to demonstrate the colocalization of Lis1 and DP. (I and J) Costaining for Ndel1 (I) or Lis1 (J) in green and β-catenin (β-cat) in red. (K and L) Staining for Ndel1 (K) and Lis1 (L) in mouse keratinocytes grown in high Ca²⁺ and treated with 10 µM nocodazole (Noc) for 1 h. DNA is labeled with Hoechst (blue) in these images. Bars, 10 µm.

Figure 2.

DP-dependent localization of Ndel1 and Lis1 in vitro and in vivo. (A–I) Localization of Ndel1 (A–C), Lis1 (D–F), and CLIP170 (G–I) in either WT keratinocytes, DP-null keratinocytes (DP KO), or α-catenin–null keratinocytes (α-cat KO), as indicated. Note the loss of cortical staining in DP-null cells. Images in A–F are maximum intensity projections of z stacks. (J and K) In vivo localization of Lis1 and Ndel1. WT (J) or DP cKO (K) e17.5 mouse skin was stained with anti-Lis1 (red) and anti–β4 integrin (green denotes the basement membrane separating dermis from epidermis). The boxed regions are magnified below the images to highlight centrosomal staining in dermis and basal cells and cortical staining in suprabasal cells. Note the loss of cortical Lis1 in the DP-null suprabasal cells. (L and M) WT (L) or DP cKO (M) e17.5 mouse skin was stained with anti-Ndel1 (red) and anti–β4 integrin (green). DNA is labeled with Hoechst (blue) in all images. Bars, 10 µm.

Figure 3.

DP recruits centrosomal proteins to the desmosome in an isoform-specific manner. (A) A diagram of the two isoforms of DP (DPI and DPII) achieved by alternative splicing. Amino acid numbers at splice sites are shown in red, and those marking domain boundaries are shown in black. (B) A table displaying DP truncation mutants. Their ability to recruit Lis1 and CLIP170, as assessed by immunofluorescence staining, is indicated. (C–H) DP knockout (KO) mouse keratinocytes were transfected with V5-tagged truncation mutants, DP 1484 (C–E), and DP 1380 (F–H). (E and H) Merged images of V5-tagged truncation mutants (green) and Lis1 (red). Images are displayed as maximum intensity projections. (I–N) DP knockout mouse keratinocytes were transfected with DPI-GFP (I) or DPII-GFP (L). Immunofluorescence staining of Lis1 in J and M. Merged images are shown in K and N. Images are maximum intensity projections. (O) WT mouse keratinocytes were cotransfected with FLAG-DP and ninein-GFP, and immunoprecipitations (IP) were performed with anti-FLAG, anti-GFP, and normal rabbit sera as a control. Bound proteins were analyzed by Western blotting. Arrows indicate the two isoforms of DP, DPI (top) and DPII (bottom). Bars, 10 µm.

Figure 4.

Lis1 is required for cortical microtubule organization in vitro and in vivo. (A–D) Immunofluorescence staining of β-tubulin in cultured keratinocytes. (A and B) Lis1 fl/fl keratinocytes not treated with Cre adenovirus. (C and D) Lis1 fl/fl keratinocytes infected with Cre adenovirus. (A and C) Control cells have predominantly cytoplasmic microtubules. (B and D) Taxol treatment induces cortical microtubules in control cells (B) but not in cells in which Lis1 has been ablated (D). A–D are maximum intensity projections of z stacks. (E and F) Sections of backskin from K14-ETMB-3GFP transgenic mice that are either WT (E) or Lis1 (F) cKO. Bars, 10 µm.

Figure 5.

Lis1-null epidermis exhibits defects in epidermal barrier function and in desmosomes. (A) Barrier assay (X-gal penetration) in Lis1 heterozygous and Lis1 cKO e18.5 embryos. Arrows indicate blisters. Bar, 1 cm. (B) Cornified envelopes from WT or Lis1 cKO mice. Bar, 40 µm. (C–N) Immunofluorescence staining of desmosomal components (as indicated on panels) on sections of backskin from WT and Lis1 cKO e18.5 embryos. The basement membrane is marked by a dashed line. Bar, 10 µm. (O) Quantitation of the cortical/cytoplasmic ratio of fluorescence intensity of DP, plakoglobin, and α-catenin (α-cat). n = 50 from each of two embryos for each genotype. Error bars represent SEM. ***, P < 0.001. (P and Q) Transmission EM images of desmosomes between two spinous layer cells in the backskin of e18.5 embryos. Arrows mark desmosomes. Bar, 0.5 µm. (R) Epidermal lysates from e18.5 WT and Lis1 cKO embryos were blotted with antibodies against the indicated proteins.

Figure 6.

Loss of Lis1 results in defects in desmosome stability. (A–J) Staining for desmosomal proteins in WT and Lis1-null keratinocytes. Images are maximum intensity projections and were taken at the same exposure settings and modified in identical ways. Bar, 10 µm. (K) Western blots of whole-cell lysates from WT, DP-null, and Lis1-null keratinocytes for the indicated proteins. Plak, plakoglobin. (L) Western blots of Lis1, epithelial cadherin (E-cad), and α-catenin (α-cat) in WT and Lis1-null cells. (M) Western blots of plakoglobin and Dsg1 from WT cells grown in either high (Hi) or low (Lo) calcium media and Lis1-null cells grown in high calcium media. (N) Levels of Dsg1 were examined from surface proteins that were biotinylated and isolated on avidin-agarose. (O and P) Turnover of desmosomal components after cycloheximide treatment. Levels of plakoglobin (O) and Dsg1 (P) were examined in WT and Lis1-null cells after treatment with cycloheximide for the indicated times. Note that initial levels of both proteins were significantly lower in Lis1-null cells than in WT cells. Error bars are SD. n = 2. (Q) A kymograph analysis of FRAP for DPI-GFP in WT cells (top) and Lis1-null cells (bottom). The bleach point is indicated. (R) Mobile fractions from individual FRAP experiments were plotted. In this box and whisker plot, the boxes represent the 25th and 75th percentiles, whereas the whiskers represent the 10th and 90th percentiles.