Single Amino Acid Deletion in Kindlin-1 Results in Partial Protein Degradation Which Can Be Rescued by Chaperone Treatment  Kristin Maier, Yinghong He, Philipp R. Esser, Kerstin Thriene, Daniela Sarca, Jürgen Kohlhase, Jörn Dengjel, Ludovic Martin, Cristina Has  Journal of Investigative Dermatology  Volume 136, Issue 5, Pages 920-929 (May 2016) DOI: 10.1016/j.jid.2015.12.039 Copyright © 2016 The Authors Terms and Conditions

Figure 1 FERMT1 mutation c.299_301del and its consequences on the skin of the patient. (a) Partial sequence of FERMT1 exon 3 in a control and patient 1. The position of the deletion mutation c.299_301del, p.R100del is indicated by a red arrow, whereas the respective codon is framed in red. (b) Immunofluorescence staining of skin sections of a healthy control, patient 1, and patient 2 with antibodies to kindlin-1 and collagen XVII. Arrow points to microblisters. Scale bars = 20 μm. (c) Immunohistochemical staining of Ki-67 in skin of the same individuals. The graph shows the quantification of the number of Ki-67 positive basal keratinocytes per 100 μm in the three individuals (control N = 16, patient 1R100del N = 6, patient 2–/– N = 9 microscopic fields). Scale bar = 100 μm. FERMT1, kindlin-1 gene. Journal of Investigative Dermatology 2016 136, 920-929DOI: (10.1016/j.jid.2015.12.039) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Molecular and functional consequences of p.R100del in keratinocytes. (a) qPCR demonstrates a 50% reduction of FERMT1 mRNA in KSKR100del compared with NHK-1. The promoter mutation in patient 2 prevents FERMT1 transcription in KSK–/–. (b) Immunoblot analyses with two distinct antibodies recognizing the N- and the C-terminus of kindlin-1 demonstrate a reduction to approximately 10% of the control levels in KSKR100del, whereas KSK–/– are completely devoid of kindlin-1. (c) Immunofluorescence staining of the three cell lines with antibodies to kindlin-1 (upper panel). Note the spikes at the periphery of NHK-1 and KSKR100del (arrow), which are absent in KSK–/–. The inset shows a twofold magnification of the marked area. Phalloidin staining (middle panel) visualized by confocal microscopy. Scale bars = 20 μm. Cells were seeded sparsely and allowed to grow for 7 days, to form well-defined colonies. Representative colonies are shown in the lower panel. Scale bar = 200 μm. (d) Quantification of cell area in a scatter plot shows a significant difference between the three cell lines (NHK-1: 1,308 μm2 ± 53.73 N = 70 cells, KSKR100del: 918.9 μm2 ± 34.75 N = 101, KSK–/–: 457.4 μm2 ± 16.73 N = 101, P < 0.0001). (e) Quantification of the number of cells per colony was performed and the mean values and SEM from three experiments are shown (N = 31, N = 30, and N = 30 colonies, respectively, P < 0.0001). FERMT1, kindlin-1 gene; KSKR100del, KS keratinocytes of patient 1 bearing the homozygous mutation p.R100del; KSK–/–, KS keratinocytes of patient 2 bearing a homozygous promoter deletion; NHK-1, normal human keratinocyte cell line 1; qPCR, quantitative real-time PCR; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 920-929DOI: (10.1016/j.jid.2015.12.039) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Low levels of wild-type and mutant kindlin-1 have similar impact on cell spreading and proliferation. (a) Immunoblot analysis of cell lysates obtained from NHK-1, KSKR100del, and KSK–/– cells, untreated or transduced with pMIG, full-length FERMT1 (+K1) or mutant cDNA (+K1R100del). Blots were incubated with antibodies to kindlin-1 and GAPDH as a loading control. (b) Cells allowed to spread were fixed and stained with phalloidin to visualize F-actin. The cell spreading area was measured using ImageJ. The graph shows a scatter plot of the results from three experiments. (c) Proliferation of NHK-1, KSKR100del, KSK–/–, KSK–/–+pMIG, KSK–/–+K1, and KSK–/–+K1R100del was assessed as described. The graph shows the average number of cells per colony and the SEM from three experiments (N = 30, N = 61, and N = 62). FERMT1, kindlin-1 gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KSKR100del, KS keratinocytes of patient 1 bearing the homozygous mutation p.R100del; KSK–/–, KS keratinocytes of patient 2 bearing a homozygous promoter deletion; KSK–/–+K1, KSK–/– cells stably expressing recombinant K1; KSK–/–+K1R100del, KSK–/– cells stably expressing recombinant K1R100del; KSK–/–+pMIG, KSK–/– cells stably transduced with pMIG; NHK-1, normal human keratinocyte cell line 1; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 920-929DOI: (10.1016/j.jid.2015.12.039) Copyright © 2016 The Authors Terms and Conditions

Figure 4 The mutation p.R100del results in misfolding and protein degradation. (a) Schematic representation of the kindlin-1 domain structure. The domains are indicated on the respective boxes. The positions of the aa, the phosphorylation sites, and the molecular interactions are indicated below. The mutation p.R100del is depicted. The framed sequence was submitted to molecular modeling. (b) Modeling of the wild-type and mutant kindlin-1 aa 1-378 is shown, and the domains are indicated as well as significant aa. The position of the mutation is circled in green (wild type) and red (K1R100del). (c) NHK-1 and KSKR100del were treated with ConA for indicated time periods. Cells were lysed and lysates submitted to immunoblot analysis with antibodies to kindlin-1 and to GAPDH. (d) Immunoblot shows kindlin-1 and GAPDH as loading control in cells that were treated with CycH for 24 hours or remained untreated. The middle framed picture represents a longer exposure of the framed area. aa, amino acid; ConA, concanamycin A; CycH, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KSKR100del, KS keratinocytes of patient 1 bearing the homozygous mutation p.R100del; NHK-1, normal human keratinocyte cell line 1. Journal of Investigative Dermatology 2016 136, 920-929DOI: (10.1016/j.jid.2015.12.039) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Effects of 4-PBA on KSKR100del. (a) NHK-1 and KSKR100del were treated with 4-PBA for the indicated time periods. NP40 soluble and insoluble fractions of cell lysates were submitted to immunoblot with antibodies to kindlin-1 and GAPDH. The lower framed pictures represent longer exposures of the upper framed areas. (b) NHK-1 and KSKR100del were treated with 4-PBA for 24 hours and stained with phalloidin. Cell area was measured as described. (c) Splicing of XBP-1 was analyzed by PCR. Amplicons were assessed by gel electrophoresis and band intensity was measured with ImageJ. The ratio of the means out of three experiments between spliced and total XBP-1 is depicted in the graph. (d) KSKR100del were treated with 4-PBA; total RNA was isolated, transcribed into cDNA, and submitted to qPCR with primers to FERMT1 and RNA18S5. FERMT1, kindlin-1 gene; KSKR100del, KS keratinocytes of patient 1 bearing the homozygous mutation p.R100del; NHK-1, normal human keratinocyte cell line 1; 4-PBA, sodium-phenylbutyrate; qPCR, quantitative real-time PCR. Journal of Investigative Dermatology 2016 136, 920-929DOI: (10.1016/j.jid.2015.12.039) Copyright © 2016 The Authors Terms and Conditions