Presentation is loading. Please wait.

Presentation is loading. Please wait.

Colocalization of Kindlin-1, Kindlin-2, and Migfilin at Keratinocyte Focal Adhesion and Relevance to the Pathophysiology of Kindler Syndrome  J.E. Lai-Cheong,

Published byIda Lesmana Modified over 5 years ago

Similar presentations

Presentation on theme: "Colocalization of Kindlin-1, Kindlin-2, and Migfilin at Keratinocyte Focal Adhesion and Relevance to the Pathophysiology of Kindler Syndrome  J.E. Lai-Cheong,"— Presentation transcript:

1 Colocalization of Kindlin-1, Kindlin-2, and Migfilin at Keratinocyte Focal Adhesion and Relevance to the Pathophysiology of Kindler Syndrome  J.E. Lai-Cheong, S. Ussar, K. Arita, I.R. Hart, J.A. McGrath  Journal of Investigative Dermatology  Volume 128, Issue 9, Pages (September 2008) DOI: /jid Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Immunoblotting shows different expression patterns of kindlin-1 (but not kindlin-2 or migfilin) in KS or control keratinocytes. Immunoblotting shows (a) ~77 and ~74kDa protein bands matching the molecular weight of kindlin-1 and its dephosphorylated form in HaCaT cell lysate (lane H). The same ~77kDa band was present in lane NHK but absent in lane KS. In addition, an ~65kDa band was seen in lane KS. (b) In lanes H, NHK, and KS, an ~77kDa band corresponding to the molecular weight of kindlin-2 was seen. (c) An ~50kDa band that equates to the expected molecular weight of migfilin was observed in lanes H, NHK, and KS. H, HaCaT cell lysate; NEG, negative control; NHK, normal human keratinocytes; KS, Kindler syndrome. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Co-immunoprecipitation studies show that kindlin-1, kindlin-2, and migfilin can bind to each other. (a) Western blotting on the immunoprecipitates using anti-kindlin-1 antibody shows ~77kDa bands in all three lanes, indicating that kindlin-1 forms a complex with kindlin-2 and migfilin. (b) Similarly, western blotting using anti-kindlin-2 antibody reveals ~77kDa bands in all three lanes, indicating that kindlin-2 associates with kindlin-1 and migfilin. (c) Immunoblotting performed using rabbit polyclonal anti-collagen VII IgG antibody as irrelevant antibody control shows absence of the ~77kDa protein band but persistence of the ~50 and ~25kDa bands. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Confocal microscopy studies show different kindlin-1 localization but preserved kindlin-2 and migfilin localization in NHK and KS keratinocytes, respectively. (a) In NHK, kindlin-1 was expressed both in the cytoplasm and near the cell periphery and colocalized with vinculin at focal adhesions. Similarly, both (b) kindlin-2 and (c) migfilin showed cytoplasmic and peripheral distribution and colocalized with vinculin. (d) In KS keratinocytes, there was cytoplasmic localization of kindlin-1 but kindlin-1 expression at focal adhesions was absent, as evidenced by the lack of colocalization with vinculin. In contrast, (e) kindlin-2 and (f) migfilin subcellular localization was not affected. Bar=20μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Confocal microscopy shows that kindlin-1, kindlin-2, and migfilin all colocalize at focal adhesions. (a) Kindlin-1 localizes to the ends of actin stress fibers and also demonstrates a perinuclear distribution. (b) Migfilin is distributed both in the cytoplasm and at the cell periphery. (c) Endogenous kindlin-2 also localizes to the ends of actin stress fibers. (d) Merged confocal image shows colocalization of kindlin-1, kindlin-2, and migfilin at focal adhesions as shown by the white staining at the cell periphery (arrows). Bar=20μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Kindlin-2 and migfilin expression is not altered after RNAi-mediated knockdown of kindlin-1 in HaCaT cells. (a) Gradual reduction of kindlin-1 expression was noted from day 2 post-transfection. (b) Expression of kindlin-2 does not seem to be altered by kindlin-1 knockdown. (c) However, there was an increase in migfilin expression from day 6 post-transfection in the scrambled control and kindlin-1-transfected HaCaT cells. Oligofectamine-only (OLI)-treated HaCaT keratinocytes lysate was used as control and showed no reduction in kindlin-1 expression. In contrast, no reduction in kindlin-1 expression was seen when the cells were treated with scrambled siRNA. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Kindlin-2 and migfilin localization remains unchanged in kindlin-1 siRNA-transfected HaCaT keratinocytes. (a) In HaCaT keratinocytes transfected with kindlin-1 siRNA, there was no localization of kindlin-1 at focal adhesions (arrows). However, (b) kindlin-2 and (c) migfilin still colocalized with vinculin at focal adhesions. In contrast, in the scrambled control-transfected HaCaT cells, there was no change in (d) kindlin-1, (e) kindlin-2, and (f) migfilin subcellular localization. Bar=20μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Immunofluorescence microscopy shows two patterns of labeling for kindlin-1, kindlin-2, and migfilin in KS skin. (a) Immunolabeling with anti-kindlin-1 antibody on normal skin sections shows bright epidermal staining predominantly at the cell periphery (but also in the cytoplasm) mostly within the basal keratinocyte layer (arrows) but with less intense pan-epidermal labeling. (b) In the KS patient with the homozygous nonsense mutation p.E516X/p.E516X there is barely detectable staining for kindlin-1. (c) In contrast, in the KS patient with the homozygous mutation p.R288X/p.R288X there is similar kindlin-1 immunolabeling intensity to control skin (cf. Figure 7a). (d) Anti-kindlin-2 immunostaining of normal skin sections shows pan-epidermal membranous labeling with the absence of staining being noted along the lower pole of basal keratinocytes. (e) Marked reduction in anti-kindlin-2 antibody immunoreactivity is seen in the patient's skin harboring the homozygous nonsense mutation p.E516X/p.E516X. (f) Kindlin-2 antibody labeling in the patient with the homozygous nonsense mutation p.R288X/p.R288X is similar to that in normal skin. (g) Anti-migfilin staining of normal skin sections shows membranous and cytoplasmic labeling of the basal keratinocytes. (h) Anti-migfilin antibody labeling in the patient with homozygous nonsense mutation p.E516X/p.E516X shows barely detectable labeling. (i) Anti-migfilin antibody labeling in the patient with the homozygous nonsense mutation p.R288X/p.R288X is similar to that in normal skin. White dashed lines indicate the position of the dermal–epidermal junction in (b), (e), and (h). Bar=50μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Semi-quantitative RT-PCR shows correlation between KIND1 mRNA expression and the corresponding kindlin-1 labeling patterns. (a) Compared with the three normal controls (lanes 1–3), patients with reduced anti-kindlin-1 immunostaining (lanes 4–6) had variably reduced KIND1 mRNA levels, whereas the patient with normal immunolabeling of anti-kindlin-1 (lane 7) had a normal or near-normal level of KIND1 mRNA. (b) Patients with reduced kindlin-2 labeling (lanes 4–6), however, had no reduction in KIND2 mRNA expression compared with normal controls (lanes 1–3) or the patient with normal kindlin-2 immunostaining (lane 7). (c) Similarly, there was no difference in FBLIM1 mRNA expression in any of the patients compared with normal controls. (d) GAPDH mRNA expression was used as loading control in these experiments. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Download ppt "Colocalization of Kindlin-1, Kindlin-2, and Migfilin at Keratinocyte Focal Adhesion and Relevance to the Pathophysiology of Kindler Syndrome  J.E. Lai-Cheong,"

Similar presentations

Nan-Hyung Kim, Ai-Young Lee  Journal of Investigative Dermatology 

Nan-Hyung Kim, Ai-Young Lee  Journal of Investigative Dermatology 
TWEAK/Fn14 Activation Contributes to the Pathogenesis of Bullous Pemphigoid  Yale Liu, Lingling Peng, Liang Li, Chengfei Liu, Xiao Hu, Shengxiang Xiao,

TWEAK/Fn14 Activation Contributes to the Pathogenesis of Bullous Pemphigoid  Yale Liu, Lingling Peng, Liang Li, Chengfei Liu, Xiao Hu, Shengxiang Xiao,
CD44 Regulates Tight-Junction Assembly and Barrier Function

CD44 Regulates Tight-Junction Assembly and Barrier Function
Volume 55, Issue 5, Pages (May 1999)

Volume 55, Issue 5, Pages (May 1999)
Complement Factor H: A Biomarker for Progression of Cutaneous Squamous Cell Carcinoma  Pilvi M. Riihilä, Liisa M. Nissinen, Risto Ala-aho, Markku Kallajoki,

Complement Factor H: A Biomarker for Progression of Cutaneous Squamous Cell Carcinoma  Pilvi M. Riihilä, Liisa M. Nissinen, Risto Ala-aho, Markku Kallajoki,
Transient Bullous Dermolysis of the Newborn Associated with Compound Heterozygosity for Recessive and Dominant COL7A1 Mutations  Nadja Hammami-Hauasli,

Transient Bullous Dermolysis of the Newborn Associated with Compound Heterozygosity for Recessive and Dominant COL7A1 Mutations  Nadja Hammami-Hauasli,
The Autoantigen in Anti-p200 Pemphigoid Is Synthesized by Keratinocytes and Fibroblasts and Is Distinct from Nidogen-2  Silke C. Hofmann, Ursula Voith,

The Autoantigen in Anti-p200 Pemphigoid Is Synthesized by Keratinocytes and Fibroblasts and Is Distinct from Nidogen-2  Silke C. Hofmann, Ursula Voith,
Role of Kindlin-2 in Fibroblast Functions: Implications for Wound Healing  Yinghong He, Philipp Esser, Vivien Schacht, Leena Bruckner-Tuderman, Cristina.

Role of Kindlin-2 in Fibroblast Functions: Implications for Wound Healing  Yinghong He, Philipp Esser, Vivien Schacht, Leena Bruckner-Tuderman, Cristina.
Basement Membrane Zone Remodeling During Appendageal Development in Human Fetal Skin. The Absence of Type VII Collagen is Associated with Gelatinase-A.

Basement Membrane Zone Remodeling During Appendageal Development in Human Fetal Skin. The Absence of Type VII Collagen is Associated with Gelatinase-A.
A Novel Component of Epidermal Cell–Matrix and Cell–Cell Contacts: Transmembrane Protein Type XIII Collagen  Sirkku Peltonen, Maria Hentula, Pasi Hägg,

A Novel Component of Epidermal Cell–Matrix and Cell–Cell Contacts: Transmembrane Protein Type XIII Collagen  Sirkku Peltonen, Maria Hentula, Pasi Hägg,
Volume 55, Issue 5, Pages (May 1999)

Volume 55, Issue 5, Pages (May 1999)
Knockdown of Myosin Va Isoforms by RNAi as a Tool to Block Melanosome Transport in Primary Human Melanocytes  Mireille Van Gele, Barbara Geusens, Anne-Marie.

Knockdown of Myosin Va Isoforms by RNAi as a Tool to Block Melanosome Transport in Primary Human Melanocytes  Mireille Van Gele, Barbara Geusens, Anne-Marie.
Protease-Activated Receptor-2 (PAR-2) Expression in Human Fibroblasts is Regulated by Growth Factors and Extracellular Matrix  Barry L. Gruber, Mary J.

Protease-Activated Receptor-2 (PAR-2) Expression in Human Fibroblasts is Regulated by Growth Factors and Extracellular Matrix  Barry L. Gruber, Mary J.
Abnormal Translocation of Tyrosinase and Tyrosinase-Related Protein 1 in Cutaneous Melanocytes of Hermansky–Pudlak Syndrome and in Melanoma Cells Transfected.

Abnormal Translocation of Tyrosinase and Tyrosinase-Related Protein 1 in Cutaneous Melanocytes of Hermansky–Pudlak Syndrome and in Melanoma Cells Transfected.
Single Amino Acid Deletion in Kindlin-1 Results in Partial Protein Degradation Which Can Be Rescued by Chaperone Treatment  Kristin Maier, Yinghong He,

Single Amino Acid Deletion in Kindlin-1 Results in Partial Protein Degradation Which Can Be Rescued by Chaperone Treatment  Kristin Maier, Yinghong He,
Hic-5 Promotes the Hypertrophic Scar Myofibroblast Phenotype by Regulating the TGF- β1 Autocrine Loop  Ganary Dabiri, David A. Tumbarello, Christopher.

Hic-5 Promotes the Hypertrophic Scar Myofibroblast Phenotype by Regulating the TGF- β1 Autocrine Loop  Ganary Dabiri, David A. Tumbarello, Christopher.
Kinesin and Kinectin Can Associate with the Melanosomal Surface and Form a Link with Microtubules in Normal Human Melanocytes1  Garnet Vancoillie, Jo.

Kinesin and Kinectin Can Associate with the Melanosomal Surface and Form a Link with Microtubules in Normal Human Melanocytes1  Garnet Vancoillie, Jo.
Upregulation of Class II β-Tubulin Expression in Differentiating Keratinocytes  Woong-Hee Lee, Joo-Young Kim, Young-Sik Kim, Hye-Joon Song, Ki-Joon Song,

Upregulation of Class II β-Tubulin Expression in Differentiating Keratinocytes  Woong-Hee Lee, Joo-Young Kim, Young-Sik Kim, Hye-Joon Song, Ki-Joon Song,
Marie-Thérèse Leccia  Journal of Investigative Dermatology 

Marie-Thérèse Leccia  Journal of Investigative Dermatology 
Regulation of IL-33 Expression by IFN-γ and Tumor Necrosis Factor-α in Normal Human Epidermal Keratinocytes  Jitlada Meephansan, Hidetoshi Tsuda, Mayumi.

Regulation of IL-33 Expression by IFN-γ and Tumor Necrosis Factor-α in Normal Human Epidermal Keratinocytes  Jitlada Meephansan, Hidetoshi Tsuda, Mayumi.

Similar presentations

Ads by Google