Crosstalk between cell adhesion and the actin cytoskeleton

In order to form tissues and to move, cells need to attach to the surrounding environment. Integrins are the major cell adhesion receptors that cells use to attach to the extracellular matrix on the outside of the cell, and to recruit a large adhesion complex on the intracellular side. As transmembrane proteins, integrins have an important role in mediating bidirectional signalling across the plasma membrane. Moreover, the integrin-based adhesions are linked to the actin cytoskeleton and thereby act as a link between the extracellular matrix and the actin cytoskeleton. The actin cytoskeleton is responsible for the cellular force generation, and integrin-based adhesions and the actin cytoskeleton create thereby a machinery, that cells can use for example to move. Moreover, integrins and the actin cytoskeleton can mediate reactions to extracellular cues and even alter gene expression. Both integrin activity and the actin cytoskeleton are carefully regulated, and mutations in genes encoding for integrin and actin regulators associate with plethora of diseases. However, less is known if integrin-based adhesions and the actin cytoskeleton are regulated by the same factors. In this thesis, I have investigated the role of two known integrin inhibitors, SHANK3 and SHARPIN, in regulation of the actin cytoskeleton, and whether this occurs synergistically with regulation of integrins and cell adhesion. I have characterised novel interaction partners for both SHANK3 and SHARPIN, and defined their functions in regulating the cellular actin cytoskeleton, cell adhesion and cell migration. Furthermore, I have investigated how SHARPIN regulates integrin activity at tissue level and find that integrin inhibition can ameliorate the effects of SHARPIN loss in vivo. Importantly, the findings presented in my thesis provide novel insights that can be used to understand pathogenesis of cancer, neuropsychiathric disorders and psoriasis-like dermatitis

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-kappa B activity. SHARPIN-deficient (Sharpin(cpdm/cpdm)) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpin(cpdm/cpdm) mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpin(cpdm/cpdm) phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1(-/-) Sharpin(cpdm/cpdm) double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpin(cpdm/cpdm) skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpin(cpdm/cpdm) mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpin(cpdm/cpdm) mice.Peer reviewe

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify signalling pathways regulated by Sharpin has not been reported. Here, we present the first ‘Sharpin interactome’, which identifies a large amount of novel potential Sharpin interactors in addition to several known ones. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with Arp2/3, a protein complex that catalyses actin filament branching. We identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin-Arp2/3 interaction promotes lamellipodium formation in a LUBAC-independent fashion.</p

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin -binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.Peer reviewe

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo

Association of the PHACTR1/EDN1 genetic locus with spontaneous coronary artery dissection

Background: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene. Objectives: This study sought to test the association between the rs9349379 genotype and SCAD. Methods: Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD. Results: The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence. Conclusions: The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD

Blocking Itgb1 activity does not inhibit inflammation in <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mice.

<p>(A, C, and E) Representative skin sections from <i>Sharpin</i>^<i>+/</i>? and <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mice treated with PBS (control) or anti-Itgb1 antibody, and stained for (A) leukocytes (CD45), (C) macrophages (F4/80) and Krt14, or (E) mast cells (Toluidine blue). Nuclei were stained with DAPI (A, C), red arrows indicate toluidine blue positive mast cells (E). Scale bars represent 20 μm (A, C) and 50 μm (E). (B, D, and F) Quantification of (B) dermal leukocyte infiltration (n = 3 or 4 animals; 10–18 measurements per animal), (D) dermal macrophage infiltration (n = 3 or 4 animals; 9–19 measurements per animal) and (F) mast cell infiltration (amount of mast cells/skin section; n = 4 or 5 animals with 10 measurements per animal) from skin sections as depicted in (A, C, and E). Numerical data are mean ± s.e.m.</p

Elevated keratinocyte apoptosis in <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mouse skin is partially rescued by Itgb1 inhibition.

<p>(A) Representative frozen skin sections from <i>Sharpin</i>^<i>+/</i>? and <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mice treated with PBS (control) or anti-Itgb1, stained for apoptotic cells [cleaved caspase-3 (Casp3)]. Red arrows indicate cleaved caspase-3 positive apoptotic cells. Scale bars represent 20 μm. (B) Quantification of epidermal apoptosis from skin sections as depicted in (A) (n = 4 <i>Sharpin</i>^<i>+/</i>? and 3 <i>Sharpin</i>^{<i>cpdm/cpdm</i>} animals; 13–18 measurements per animal). Numerical data are mean ± s.e.m.</p

SHANK3 in oncogenic RAS signaling.

peer reviewe

Keratinocytes from <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} double knockout mice have higher levels of active Itgb1.

<p>(A) Representative frozen skin sections from 6 weeks old <i>Tnfr1</i>^<i>+/+</i> <i>Sharpin</i>^<i>+/</i>?, <i>Tnfr1</i>^<i>+/+</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>}, <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^<i>+/</i>? and <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mice (n = 3 animals per genotype) stained for Keratin-14 (Krt14) and active Itgb1 (clone 9EG7). Scale bars represent 50 μm. Arrows indicate the basal cell layer. (B) Representative frozen skin sections from 6 weeks old <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mice stained for Krt14 (epidermis) and Itga6 (basal keratinocytes). Scale bars represent 10 μm. (C) Primary keratinocytes were isolated from <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^<i>+/</i>?, <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} and <i>Tnfr1</i>^<i>+/+</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} mice and studied by flow cytometry. Live cells (FSC/SSC; left panel) were sorted for expression of Itga6 (CD49f) and lineage (Lin) markers CD31 and CD45 (central panel). Cell surface expression of active Itgb1 (CD29; clone 9EG7) was plotted (right panel) from the gated basal keratinocyte population (live, Itga6⁺Lin^-). (D,E) Relative geometric mean fluorescence intensities of (<b>D</b>) active Itgb1 (n = 6, 5 and 4 animals per genotype) and (E) total Itgb1 (n = 4, 4 and 3 animals per genotype) cell surface staining on <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^<i>+/</i>?, <i>Tnfr1</i>^<i>-/-</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} and <i>Tnfr1</i>^<i>+/+</i> <i>Sharpin</i>^{<i>cpdm/cpdm</i>} basal keratinocytes sorted as in (<b>C</b>). Numerical data are mean ± s.e.m.</p