Integrin activity regulation in mechanosensing: Integrin beta1 activity and extracellular matrix crosstalk

Cell functions are highly dependent on the physical properties of the extracellular matrix (ECM). Integrins are transmembrane receptors providing the main molecular link that attaches cells to the ECM. The distinct biochemical and mechanical properties of integrins enable them to act as bi-directional signalling-hubs. By probing the ECM stiffness via integrin adhesions, cells respond to the rigidity of their environment by tuning proliferation, gene expression and even their epigenetic landscape. Furthermore, integrin adhesions allow cells to exert actomyosingenerated force on the ECM, enabling processes such as cell migration and ECM remodelling. Consequently, integrin activity needs to be thoroughly regulated on several levels. While the role of integrins as key cellular mechanosensors is well established, the link between mechanosensing and integrin activity regulation by integrin inactivators and newly synthesized integrins, is poorly understood. In this thesis, I have identified the integrin activity inhibitor SHARPIN as an important regulator of ECM remodelling in the developing mammary gland. By regulating the levels of the collagen-binding integrin α11β1, SHARPIN affects the force generation capacity of mammary gland stromal fibroblasts and thereby their ability to remodel and assemble the ECM to support normal mammary gland development. Furthermore, I have investigated the trafficking and localization of newly synthesized integrin α5β1. My results show that newly synthesized integrins are trafficked in a polarized manner to the tip of adhesions where they contribute to adhesion growth in a ligand-dependent manner. In addition, we find that a subset of newly synthesized integrin α5 can undergo unconventional secretion, to be rapidly trafficked to cell adhesions. Together, these results provide novel insights of how the crosstalk between the ECM and integrin activity regulates cell behaviour, and may be essential in understanding the pathological cell migration and ECM remodeling events in cancer.Integriinien aktiivisuus säätely mekanotunnustelussa Soluväliaineen fyysiset ominaisuudet vaikuttavat eri solutoimintoihin, kuten solun liikkumiseen ja solunjakautumiseen. Integriinit läpäisevät solukalvon, tarjoavat soluille mekanismin sitoutua soluväliaineeseen ja toimivat kaksisuuntaisina signaalinvälittäjinä. Tunnustelemalla soluväliaineen jäykkyyttä integriinien kautta, solut reagoivat jäykkyyteen säätelemällä solunjakautumista, geenien ilmentymistä ja jopa epigeneettistä tilaa. Integriini-adheesiot välittävät myös solujen aktiinitukirangan tuottamat voimat soluväliaineeseen, täten mahdollistaen toimintoja kuten solujen liikkumista ja soluväliaineen uudelleenmuokkaamista. Integriinien aktiivisuus ja siitä riippuvainen voimansiirto ovat tästä johtuen olennaisia solutoiminnoissa, jotka liittyvät sekä yksilönkehitykseen että erilaisiin sairauksiin. Tästä syystä, integriinien aktiivisuus on tarkasti säädeltyä monella eri tasolla. Vaikka integriinien toiminta mekaanisina antureina tunnetaan hyvin, voimansiirron ja integriinien aktiivisuusäätelyn välinen yhteys tunnetaan huonosti. Tässä väitöskirjatutkimuksessa olen tunnistanut integriiniaktiivisuusinhibiittorin SHARPIN:in tärkeänä säätelijänä soluväliaineen muokkaamisessa rintarauhasen kehityksessä. Säätelemällä kollageenia sitovan integriini α11β1 tasoja, SHARPIN vaikuttaa rintarauhasen tukikudossolujen voimaansiirtokapasiteettiin ja täten niiden kykyyn muokata soluväliainetta kehittyvässä rintarauhasessa. Lisäksi olen tutkinut vasta tuotettujen integriini α5β1-reseptorien kalvokuljetusta ja kohdentamista soluissa. Tulokseni osoittavat, että vasta tuotettujen integriinien kalvokuljetus on polarisoitunutta siten, että nämä integriinit lokalisoituvat adheesioiden päihin, edesauttaen adheesioiden kasvua ligandi-riippuvaisella tavalla. Osa vastatuotetuista integriineistä erittyy myös epätavanomaisesti solun pinnalle ohittaen Golgi-välitteisen erityksen kulkeutuen nopeasti adheesioihin. Väitöskirjani löydökset tarjoavat uutta tietoa integriinien ja soluväliaineen vuorovaikutuksesta ja näiden osuudesta solutoiminnan säätelyssä. Nämä löydökset saattavat auttaa paremmin ymmärtämään syövässä esiintyviä tautimekanismeja, kuten soluliikkumista ja soluväliaineen muokkaamista

Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells

Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types, and may also replace or repair diseased brain tissue. NSCs can be directly isolated from fetal or adult nervous tissue, or derived from embryonic stem cells. Here, we describe the efficient conversion of human adult bone marrow stromal cells (hMSC) into a neural stem cell-like population (hmNSC, for human marrow-derived NSC-like cells). These cells grow in neurosphere-like structures, express high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, MSl1 as well as otx1 and nestin, but lose the characteristics of mesodermal stromal cells. In the presence of selected growth factors, hmNSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Clonal analysis demonstrates that individual hmNSCs are multipotent and retain the capacity to generate both glia and neurons. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.Peer reviewe

Targeting beta 1-integrin inhibits vascular leakage in endotoxemia

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against beta 1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. beta 1-integrin inhibiting antibodies bound to the vascular endotheliumin vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial beta 1-integrin protected mice from LPS-induced vascular leakage. In endothelial mono-layers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1 beta decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via beta 1- and alpha 5-integrins and ANGPT2. Additionally, beta 1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of alpha 5 beta 1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, beta 1-integrin promotes endothelial barrier disruption during inflammation, and targeting beta 1-integrin signaling could serve as a novel means of blocking pathological vascular leak.Peer reviewe

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts

The link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.publishedVersio

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.</p

AMPK negatively regulates tensin-dependent integrin activity

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a beta 1-integrin inhibitor in fibroblasts. Loss of AMPK promotes beta 1-integrin activity, the formation of centrally located active beta 1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases beta 1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind beta 1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.Peer reviewe

Democratising deep learning for microscopy with ZeroCostDL4Mic

Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes. Deep learning methods show great promise for the analysis of microscopy images but there is currently an accessibility barrier to many users. Here the authors report a convenient entry-level deep learning platform that can be used at no cost: ZeroCostDL4Mic

16p11.2 600 kb Duplications confer risk for typical and atypical Rolandic epilepsy

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65 046 European population controls (5/393 cases versus 32/65 046 controls; Fisher's exact test P = 2.83 × 10−6, odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10−4). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical R