Kindlin and EGFR control convergent pathways to regulate epithelial cell function

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

This research relates to two proteins that we know are not correctly controlled in patients with skin blistering disease. These proteins are called Kindlin-1 and Epidermal Growth Factor Receptor (or EGFR). We already know that lack of Kindlin-1 in the skin leads to Kindler Syndrome - a rare genetic disorder that results in skin blistering and sometimes leads to skin cancer. We have also recently identified a new group of patients that have defective EGFR protein and this similarly results in fragile skin. Our recent research on these two proteins strongly suggests that the observed clinical defects in the patients are partly caused by similar processes going wrong. This defect is mainly in the ability of cells to stick, or 'adhere' to the proteins that surround them in the skin. Normal functioning of the adhesion process is critical to health, and if it goes wrong, the sort of diseases that might occur include skin fragility, psoriasis, cancer, reduced immunity and inflammation. The research we would like to do now involves analysing Kindlin-1 and EGFR function in more detail. We firstly want to know precisely how these proteins work together to contribute to normal skin function. We plan to use a number of biochemical and microscopy techniques to answer questions such as: do these two proteins bind together in cells; how does loss of one or the other upset the ability of cells to adhere to proteins around them, and how do the common 'targets' of this protein complex also control this process? In our research we would also like to create more complex models to allow us to study Kindlin-1 and EGFR function in an intact organism. The reason we want to do this is because we would like to find out how these proteins behave in a more 'real' setting where there are other cells and tissues that might alter how these proteins behave. We will use a very well characterized model of epidermal blistering in the Fruitfly for this purpose. These animals are much easier, cheaper and faster to work with than mice and have versions of all of the genes/proteins we are planning to study, so are ideal for our experiments. By combining the information we get from experiments in cells, human tissues and the Fruitfly model, we can also start to plan potential corrective treatments with genes, proteins and drugs, work that could lead to improvements for patients in the future. Such work could lead to less skin blistering and improved quality of life in this and other families that have defects in Kindlin-1 or EGFR. The work is expected to expand our knowledge of the fundamentally important basic science topic of cell adhesion to surrounding tissues as well as the physiological maintenance of healthy skin.

Technical Summary

The goal of this project is to integrate the identification of new mutations in patients with skin blistering with a cell biological analysis of the defects in mammalian cells, and exploitation of the genetics of Drosophila to group components into novel pathways. Our serendipitous findings to date provide us with a unique opportunity to interrogate the co-operation and convergent molecular signatures of EGFR and kindlin-1-dependent skin disease. Our first goal is to confirm that Kindlin-1 regulation of EGFR is a key part of its unique function that cannot be substituted for by Kindlin-2. Second, we wish to identify the mechanism by which Kindlin-1 regulates EGFR levels. Third, we aim to identify the common mediators of targets of EGFR ad Kindlin-1 signalling that mediate the epithelial cell adhesion. These studies will characterise novel candidate genes and mechanisms that contribute to skin fragility disorders using a powerful combination of in vitro and in vivo models: human keratinocytes (from healthy volunteers and KS patients) and the genetically tractable organism Drosophila. We anticipate that the knowledge gained will improve our understanding of how changes in levels or activation of each of the pathway components gives rise to the disease pathology, suggests possible treatments, and aids in the identification of less serious skin conditions that may arise from a more mild impairment of these pathways.

Planned Impact

Data arising from this study will be of significant interest to a wide range of cell and developmental biologists. The questions we are approaching in this study are of immediate relevance to researchers in the cell adhesion and migration field, and adhesion/growth factor receptor crosstalk is well recognized as being centrally important to a wide range of basic biological processes such as proliferation and migration. Adhesion and growth factor signalling are evolutionarily conserved events and therefore our data stand to benefit a wide collection of investigators in basic and translational research, including biologists, microscopists, immunologists, infectious disease specialists, neurobiologists, and oncologists. The Kindlin proteins are currently emerging as key players in other disease settings including cancer, immune dysfunction and wound healing. Moreover, EGFR is well-known to be dysregulated in many cancers so provision of novel information relating to potential upstream or downstream regulators will help inform on potential combined treatments in EGFR mutated disease settings. The outcomes will also be important to clinical dermatologists both from a diagnostic and treatment perspective, as unraveling these pathways provides a platform for future translational research including gene, protein, cell and drug therapies aimed at restoring function in patient skin and reducing the skin blistering.
The collaboration between basic cell and developmental biology and clinical researchers proposed here provides an exciting and powerful multidisciplinary team to dissect the full implications of Kindlin-EGFR signaling in the context of epithelial homeostasis and disease. The workplan offers excellent training opportunities for the new team members and provides a platform for open discussions between all three labs that will not only benefit the current study but likely lead to future collaborative projects together. Moreover, our novel discoveries and related studies are expected to open the door to new collaborations with both European and global investigative research teams over the next 1-3 years.
Outside the immediate academic beneficiaries, data obtained in this study will also be of benefit to the patient charitable organisation that assists individuals with inherited blistering skin diseases. This organisation is called the Dystrophic Epidermolysis Bullosa Research Association (DebRA) and it provides support for more than 5,000 people in the UK with different forms of genetic blistering diseases involving mutations in 14 different genes. The data arising from this study will also be of direct benefit to the NHS diagnostic laboratory for epidermolysis bullosa based at St Thomas' Hospital in London. This laboratory (The Robin Eady National Diagnostic Epidermolysis Bullosa Laboratory) will now be able to add additional diagnostic tests (immunohistochemistry for EGFR, FRMPD1, UNC93, mutation analysis of associated genes) to make its diagnostic services even more comprehensive and inclusive.
 
Title Additional file 1 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 1: Figure S1. (a) Images of WT monolayers with 2mM Ca2+ fixed and stained for F-actin and a3, a3 or a5 integrin subunits and ß1 integrin. Scale bars, 10µm. (b) Images of WT monolayers +/- 2mM Ca2+ fixed and stained for F-actin and active ß1 integrin. Quantification of relative junctional intensities of total and active ß1 integrin without and with 2mM Ca2+ from 35 cells per condition; representative of 3 independent experiments. Scale bars, 10µm. (c) Images of WT monolayers with 2mM Mg2+ +/- Ca2+ fixed and stained for F-actin and active ß1 integrin. Quantification of relative junctional intensities of active ß1 integrin without and with 2mM Mg2+ or Ca2+ from 30 cells per condition; representative of 3 independent experiments. Scale bars, 10µm. (d) Western blot analysis of cells for integrin subunits a2, a3, a5 and ß1 or actin, E-cadherin and ß-catenin with GAPDH as a loading control. (e) Representative confocal images of Control and a2KD cells or WT cells treated with DMSO or BT fixed and stained for F-actin and active ß1 integrin. Scale bars, 10µm. (f) Representative confocal images of WT cells treated with DMSO or BTT (20 µm, 1hr) fixed and stained for F-actin and E-cadherin and quantification of E-Cadherin and F-actin intensity at junctions from 30 cells per condition from 3 independent experiments. Scale bars 10µm. (g) Confocal slices from junctional and basal planes of Control and a2KD monolayers in 2mM Ca2+ fixed and stained for F-actin and vinculin and quantification of vinculin positive focal adhesion at basal planes from 35 cells per condition; representative of 3 independent experiments. Scale bars, 10µm. (h) Analysis of cell monolayer permeability in WT, Control, a2 knockdown (KD1 and KD2) and a2KD1 cells re-expressing a2-GFP following 2 hours of fluorescent dextran incubation. 1mM EDTA was used a positive control. Data is from n=4 wells per condition, and representative of 3 independent experiments. (i) Analysis of proliferation of WT, Control and a2 knockdown (KD1 and KD2) and KD1 cells stably rescued with a2-GFP over 72h under normal growth conditions. n=4 wells per condition; representative of 3 independent experiments. (j) Quantification of % wound closure from 24h movies of WT, Control, a2 knockdown (KD1 and KD2) and a2KD1 cells re-expressing a2-GFP. n=3 wells per condition; representative of 3 independent experiments. *** p<0.001, **p<0.01, *p<0.05. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_1_of_2_1_integrins_spatially_res...
 
Title Additional file 1 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 1: Figure S1. (a) Images of WT monolayers with 2mM Ca2+ fixed and stained for F-actin and a3, a3 or a5 integrin subunits and ß1 integrin. Scale bars, 10µm. (b) Images of WT monolayers +/- 2mM Ca2+ fixed and stained for F-actin and active ß1 integrin. Quantification of relative junctional intensities of total and active ß1 integrin without and with 2mM Ca2+ from 35 cells per condition; representative of 3 independent experiments. Scale bars, 10µm. (c) Images of WT monolayers with 2mM Mg2+ +/- Ca2+ fixed and stained for F-actin and active ß1 integrin. Quantification of relative junctional intensities of active ß1 integrin without and with 2mM Mg2+ or Ca2+ from 30 cells per condition; representative of 3 independent experiments. Scale bars, 10µm. (d) Western blot analysis of cells for integrin subunits a2, a3, a5 and ß1 or actin, E-cadherin and ß-catenin with GAPDH as a loading control. (e) Representative confocal images of Control and a2KD cells or WT cells treated with DMSO or BT fixed and stained for F-actin and active ß1 integrin. Scale bars, 10µm. (f) Representative confocal images of WT cells treated with DMSO or BTT (20 µm, 1hr) fixed and stained for F-actin and E-cadherin and quantification of E-Cadherin and F-actin intensity at junctions from 30 cells per condition from 3 independent experiments. Scale bars 10µm. (g) Confocal slices from junctional and basal planes of Control and a2KD monolayers in 2mM Ca2+ fixed and stained for F-actin and vinculin and quantification of vinculin positive focal adhesion at basal planes from 35 cells per condition; representative of 3 independent experiments. Scale bars, 10µm. (h) Analysis of cell monolayer permeability in WT, Control, a2 knockdown (KD1 and KD2) and a2KD1 cells re-expressing a2-GFP following 2 hours of fluorescent dextran incubation. 1mM EDTA was used a positive control. Data is from n=4 wells per condition, and representative of 3 independent experiments. (i) Analysis of proliferation of WT, Control and a2 knockdown (KD1 and KD2) and KD1 cells stably rescued with a2-GFP over 72h under normal growth conditions. n=4 wells per condition; representative of 3 independent experiments. (j) Quantification of % wound closure from 24h movies of WT, Control, a2 knockdown (KD1 and KD2) and a2KD1 cells re-expressing a2-GFP. n=3 wells per condition; representative of 3 independent experiments. *** p<0.001, **p<0.01, *p<0.05. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_1_of_2_1_integrins_spatially_res...
 
Title Additional file 2 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 2: Figure S2. a) Quantification of the percentage of cells adhered onto collagen, LN or Fc-E-cadherin following 60 minutes incubation, representative of 3 independent experiments. (b) Representative image of control cells plated onto Fc-E-cadherin coated coverslip for 30 minutes and fixed and stained for a2 integrin and E-cadherin. Scale bar 10µm. (c) Confocal images of basal plane of WT monolayers in 2mM Ca2+, fixed and stained for DAPI, laminin a3 and F-actin. Scale bars 10µm. *** p<0.001, *p<0.05. (d) Representative confocal images of human skin sections stained for a2 integrin, laminin a3, Laminin ß1 or Collagen IV. Bottom panel shows zoomed images of example regions where Laminin interdigitates between keratinocytes. Scale bars 10µm. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_2_of_2_1_integrins_spatially_res...
 
Title Additional file 2 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 2: Figure S2. a) Quantification of the percentage of cells adhered onto collagen, LN or Fc-E-cadherin following 60 minutes incubation, representative of 3 independent experiments. (b) Representative image of control cells plated onto Fc-E-cadherin coated coverslip for 30 minutes and fixed and stained for a2 integrin and E-cadherin. Scale bar 10µm. (c) Confocal images of basal plane of WT monolayers in 2mM Ca2+, fixed and stained for DAPI, laminin a3 and F-actin. Scale bars 10µm. *** p<0.001, *p<0.05. (d) Representative confocal images of human skin sections stained for a2 integrin, laminin a3, Laminin ß1 or Collagen IV. Bottom panel shows zoomed images of example regions where Laminin interdigitates between keratinocytes. Scale bars 10µm. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_2_of_2_1_integrins_spatially_res...
 
Title Additional file 3 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 3: Figure S3. (a) Images of control and a2 knockdown (KD) cells treated with either DMSO or ML141 (10 µm, 4h) and fixed and stained for DAPI and E-cadherin. Scale bars, 10µm. (b) Quantification of E-cadherin intensity at junctions and junction width from images as in (a). (c) Representative blots of lysates from a2KD cells expressing GFP or a2-GFP with or without 2mM Ca2+ (- and + respectively), immunoprecipitated with GFP antibodies and complexes probed for a2, Cdc42 or GFP. Input levels are shown on the left. (d) Representative blots of lysates from a2KD cells expressing GFP or a2-GFP with or without 2mM Ca2+ (- and + respectively), immunoprecipitated with GFP antibodies and complexes probed for a2, IQGAP1, RhoGDI, RacGAP1 or Tuba. Input levels are shown on the left. (e) Images of DMSO and BTT treated cells fixed and stained for pY156 RhoGDI and E-Cadherin; quantification of images from at least 30 images per condition over 3 independent experiments. Scale bars, 10µm. (f) GFP trap of lysates from WT cells expressing either GFP or RhoGDIa-GFP treated with DMSO or PP2 (10 µm, 1hr). Complexes from GFP traps were probed for phosphotyrosine (PY) and GFP. (g) GFP trap of lysates from WT cells expressing either GFP or RhoGDIa-GFP treated with Ca2+ (2mM) for 5 mins. Complexes from GFP traps were probed for phosphotyrosine (PY) and GFP. ***= p<0.001, **= p<0.01, *= p<0.05. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_3_of_2_1_integrins_spatially_res...
 
Title Additional file 3 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 3: Figure S3. (a) Images of control and a2 knockdown (KD) cells treated with either DMSO or ML141 (10 µm, 4h) and fixed and stained for DAPI and E-cadherin. Scale bars, 10µm. (b) Quantification of E-cadherin intensity at junctions and junction width from images as in (a). (c) Representative blots of lysates from a2KD cells expressing GFP or a2-GFP with or without 2mM Ca2+ (- and + respectively), immunoprecipitated with GFP antibodies and complexes probed for a2, Cdc42 or GFP. Input levels are shown on the left. (d) Representative blots of lysates from a2KD cells expressing GFP or a2-GFP with or without 2mM Ca2+ (- and + respectively), immunoprecipitated with GFP antibodies and complexes probed for a2, IQGAP1, RhoGDI, RacGAP1 or Tuba. Input levels are shown on the left. (e) Images of DMSO and BTT treated cells fixed and stained for pY156 RhoGDI and E-Cadherin; quantification of images from at least 30 images per condition over 3 independent experiments. Scale bars, 10µm. (f) GFP trap of lysates from WT cells expressing either GFP or RhoGDIa-GFP treated with DMSO or PP2 (10 µm, 1hr). Complexes from GFP traps were probed for phosphotyrosine (PY) and GFP. (g) GFP trap of lysates from WT cells expressing either GFP or RhoGDIa-GFP treated with Ca2+ (2mM) for 5 mins. Complexes from GFP traps were probed for phosphotyrosine (PY) and GFP. ***= p<0.001, **= p<0.01, *= p<0.05. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_3_of_2_1_integrins_spatially_res...
 
Title Additional file 4 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 4: Figure S4. (a) Images of Control and a2 knockdown (KD) monolayers in Ca2+, fixed and stained for p-Src and E-cadherin. Scale bars 10µm. (b) Images of Control and a2 knockdown (KD) monolayers in Ca2+, treated with either DMSO or PP2 (10 µm, 1hr), fixed and stained for E-cadherin. Scale bars 10µm. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_4_of_2_1_integrins_spatially_res...
 
Title Additional file 4 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 4: Figure S4. (a) Images of Control and a2 knockdown (KD) monolayers in Ca2+, fixed and stained for p-Src and E-cadherin. Scale bars 10µm. (b) Images of Control and a2 knockdown (KD) monolayers in Ca2+, treated with either DMSO or PP2 (10 µm, 1hr), fixed and stained for E-cadherin. Scale bars 10µm. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_4_of_2_1_integrins_spatially_res...
 
Title Additional file 5 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 5. Full blots for all data shown in Figs. 1, 2, 3, 4 and Additional Files 1, 2, 3, 4. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_5_of_2_1_integrins_spatially_res...
 
Title Additional file 5 of a2ß1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions 
Description Additional file 5. Full blots for all data shown in Figs. 1, 2, 3, 4 and Additional Files 1, 2, 3, 4. 
Type Of Art Film/Video/Animation 
Year Produced 2021 
URL https://springernature.figshare.com/articles/figure/Additional_file_5_of_2_1_integrins_spatially_res...
 
Description Defining mechanism of action of wound healing compounds
Amount £235,000 (GBP)
Organisation Amryt Pharma 
Sector Private
Country Germany
Start 05/2020 
End 06/2021
 
Description Mechanisms controlling integrated cell adhesion
Amount £282,179 (GBP)
Funding ID RPG-2021-361 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2022 
End 04/2024
 
Description Collaboration on structural biology of kindlin 
Organisation University of Kent
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided new data to now be analysed by our collaborator using structural approaches
Collaborator Contribution Our collaborators will analyse a novel protein-protein interaction using advanced biophysical methods
Impact Multi-disciplinary -no outcomes yet, still in progress
Start Year 2016
 
Description Research collaboration with Amryt Pharma 
Organisation Amryt Pharma
Country Germany 
Sector Private 
PI Contribution This is a new collaboration to identify the mechanism of action of an agent currently in clinical trials to treat epidermolysis bullosa
Collaborator Contribution Our partners (Amryt) have developed an agent for the treatment of epidermolysis bullosa which is currently in PhaseIII clinical trials
Impact Collaboration due to commence March 2020 so not outputs yet
Start Year 2020
 
Description Presentation at Actin 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presentation of unpublished data - sparked new discussion with other leaders in Actin field
Year(s) Of Engagement Activity 2016
 
Description Talk at GRC Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation of data to other peers and experts in the field - generated significant interest and established new collaborations
Year(s) Of Engagement Activity 2016