Carcinona epidermóide é um prevalente tumor de cabeça e pescoço relacionado a altas taxas de mortalidade. Neste trabalho, verificamos se AG73 (RKRLQVQLSIRT, cadeia a1), peptídeo derivado da laminina-111, regula migração, invasão e secreção de protease em células de carcinoma epidermóide oral (OSCC). Cadeia a1 da laminina e MMP9 estão expressas neste tumor in vivo e in vitro. AG73 induziu aumento da taxa migratória de células OSCC em ensaios de ferida e migração, e também estimulou invasão em ensaio em câmaras bipartites com Matrigel. Células OSCC crescidas sobre AG73 exibiram aumento dose-dependente de MMP9, detectado por zimografia. Buscamos receptores de AG73 que regulariam atividade nesta linhagem. Células OSCC crescidas sobre AG73 exibiram colocalização de sindecana-1 e integrina b1, e silenciamento desses receptores com RNA de interferência promoveu diminuição de migração e invasão dependente de AG73 nestas células. Esses resultados sugerem que sindecana-1 e integrina b1, ativados por AG73, podem regular migração, invasão e secreção de MMPs em células OSCC. / Oral squamous cell carcinoma is a prevalent head and neck tumor, related to high mortality rates. Here we studied the role played by AG73 (RKRLQVQLSIRT, a1 chain) on migration, invasion and protease secretion of a cell line (OSCC) from human oral squamous cell carcinoma. Laminin a1 chain and MMP9 are expressed in oral squamous cell carcinoma cells in vivo and in vitro. AG73 increased migratory activity of OSCC cells, as shown by monolayer wound assays and migration assays. This peptide also stimulated cell invasion in chemotaxis chambers coated with Matrigel. OSCC cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, detected by zymography. We searched for AG73 receptors regulating activities in this cell line. OSCC cells grown on AG73 exhibited colocalization of syndecan-1 and b1 integrin, and siRNA knockdown of these receptors decreased AG73-dependent migration and invasion of OSCC cells. Our results suggest that syndecan-1 and b1 integrin signaling downstream of AG73 regulate migration, invasion and MMP secretion by OSCC cells

Estudamos indução da atividade de MMP pelo peptídeo da laminina a1 AG73 em linhagem celular (CAC2) de carcinoma adenóide cístico. CAC2 cultivadas em laminina-111 com AG73 geraram espaços pseudocísticos. Inibidor de MMP diminuiu tais espaços, sugerindo ação de MMPs. CAC2 crescidas sobre AG73 mostraram aumento dose-dependente de MMP9. RNAi para MMP9 diminuiu remodelação em cultura 3D. Buscamos receptores de AG73 ligados à atividade de MMP9. CAC2 crescidas sobre AG73 exibiram colocalização de sindecan-1 e integrina b1. RNAi para sindecan-1 ou para integrina b1 geraram, isolados, redução na adesão a AG73 e nas atividades de remodelação e de protease. Duplo RNAi estudou a cooperação entre os receptores e promoveu diminuição na adesão a AG73 e na atividade de MMP. Distinção de receptores foi feita por cromatografia de afinidade e espectrometria de massa, através de colunas de afinidade com AG73 acoplado, que resultou em possíveis receptores, como integrinas b1 e aV. Sugerimos que AG73 regula adesão e secreção de MMP em células CAC2 através de sindecan-1 e integrina b1. / We studied induction of MMP activity by b1-laminin peptide AG73 in adenoid cystic carcinoma cell line (CAC2). Cells grown inside AG73-enriched laminin-111 exhibited pseudocystics spaces. MMP inhibitor decreased those spaces, suggesting MMPs action. Cells grown on AG73 showed a dose-dependent increase of MMP9 secretion. MMP9 siRNAi decreased remodeling in 3D culture. We searched for AG73 receptors regulating MMP9 activity. CAC2 grown on AG73 exhibited colocalization of syndecan-1 and b1 integrin. Syndecan-1 siRNA or siRNA b1 integrin showed reduction in adhesion to AG73 and in remodeling and protease activities. Double-knockdown explored syndecan-1 and 1 integrin cooperation and showed decrease in adhesion to AG73 and in MMP activity. Receptors characterization was made by affinity chromatography followed by mass spectrometry through AG73-affinity columns and showed putative receptors, like b1 and aV integrins. We suggest that AG73 peptide regulates adhesion and MMP secretion in CAC2 cells through syndecan-1 and b1 integrin.

La mucopolysaccharidose de type IIIB (MPSIIIB), ou maladie de Sanfilippo B, est une maladie de surcharge lysosomale caractérisée par des atteintes neurologiques. Cette maladie génétique rare est causée par la déficience en a-N-acétylglucosaminidase (NAGLU), une enzyme nécessaire pour la dégradation des héparanes sulfates (HS). La dégradation incomplète des HS cause l’accumulation de saccharides d’HS dans les lysosomes et à la surface des cellules. Mais la cascade physiopathologique induite par ces saccharides n’est pour l’instant pas connue. D’une part, ces recherches fournissent des preuves que la communication avec l’environnement des cellules neurales déficientes en NAGLU est altérée. En effet, l’intégrine ß1 et ses effecteurs sont suractivés et recrutés au niveau des plaques d’adhérence dans des astrocytes déficients. Les comportements cellulaires dépendants des intégrines, tels que la polarisation et la migration, sont également altérés. Ces phénotypes sont restaurés par l’apport de l’enzyme déficiente. Cette restauration indique que l’accumulation de saccharides d’HS provoque l’activation de la signalisation des intégrines, et perturbe la polarisation et la migration des cellules neurales. L’ajout de saccharides d’HS purifiés sur des cellules neurales normales confirme que les saccharides d’HS extracellulaires activent des composants des plaques d’adhérence. D’autre part, l’étude d’un modèle cellulaire humain, dont l’expression de NAGLU a été inhibée par shRNA, a montré que l’accumulation de vésicules de stockage caractéristiques de la maladie est causée, entre autre, par une déformation de l’appareil de Golgi et la surexpression de GM130. Ces phénotypes sont également observés dans les neurones atteints. Ils s’accompagnent d’une augmentation de la stabilité et de la nucléation des microtubules, au niveau de l’appareil de Golgi. Les défauts de communication entre la cellule malade et son environnement semblent donc modifier la dynamique et la structure cellulaire. Nous présumons que les mécanismes physiopathologiques déchiffrés en culture sont reliés à la neuropathologie de la MPSIIIB. En perturbant la perception de l’environnement cellulaire, la polarité, la migration, et la pousse neuritique, les saccharides d’HS accumulés dans les tissus cérébraux malades, affectent probablement divers mécanismes clefs de la maturation corticale. / Mucopolysaccharidosis type IIIB (Sanfilippo B disease) is a lysosomal storage disease characterized by severe neurological manifestations in children. This rare monogenic disease is caused by a-N­acetylglucosaminidase (NAGLU) deficiency, a lysosomal hydrolase necessary for heparan sulfate (HS) degradation. This deficiency leads to the accumulation of HS saccharides. Mechanisms mediating HS saccharides deleterious effects on brain cells are not well understood. This research provides evidences that neural cell sensing of environment is altered in MPSIIIB cells. Integrins and focal adhesion components are over-recruited and over-activated in deficient mouse astrocytes. Consistently, integrin-dependant cell behavior such as cell polarization and directed migration were defective in affected astrocytes and neural stem cells. HS saccharide clearance, by NAGLU gene transfer, rescues a normal phenotype suggesting that HS saccharides induce focal adhesion formation. Addition of purified HS saccharides on normal astrocytes confirms that extracellular HS saccharides can activate the recruitment of focal adhesion components and provides an in vitro assay to decipher the saccharide code of HS. Otherwise, investigations performed on HeLa cell model, in which NAGLU expression was inhibited by shRNA, showed that accumulation of intracellular storage vesicles, a hallmark of the disease, is due over expression of a cis-Golgi protein. This affects the Golgi morphology and microtubule nucleation and stability. It seems that alterations of environment cell sensing and downstream signaling also modify the dynamic and the structure of cells. We assume that mechanisms deciphered in cell cultures are related to MPSIIIB neuropathology. By affecting cell perception of environmental cues, cell polarity, cell migration and neurite outgrowth, HS saccharides, which accumulate in brain tissues defective for a HS degradation enzyme, likely affect various processes important for accurate cortical maturation.

Adhesion between the plasma membrane and the cell wall and the existence of the continuum of these two compartments is needed for signal transmission, e.g. under pathogen attack, during cell expansion and cell wall growth, or in response to environmental conditions. This adhesion is, in addition to the turgor, provided by physical connection of both compartments. One of the best-known examples of physical connection is found in the root system, in the Caspary strip region, where it is required to maintain apoplastic barriers of the root system, even under adverse conditions and consequent plasmolysis. There is little information about the physical interconnection and the participating macromolecules but there are candidates, which could participate in this interaction. The diploma thesis deals with arabinogalactan (AGP) proteins with fasciclin-like domain (namely FLA9 and FLA2). These proteins may play a role in the adhesion of plasma membrane and cell wall and may be involved in the growth regulation and development of the root system. Both genes are are relatively strongly expressed in the roots, especially in the elongation zone and in the cortex, including the endodermis (according to chip data). Also, to a smaller extent, the thesis deals with integrin-like At14A protein. The most interesting...

Le CD154, est une glycoprotéine transmembranaire de type II appartenant à la famille des TNF (tumor necrosis factor). Il est exprimé de façon inductible et transitoire sur plusieurs types cellulaires. Il existe deux formes du CD154, membranaire (mCD154) et soluble (sCD154) qui forment une structure trimérique biologiquement active. Le CD154 a été initialement identifié pour son rôle important dans la réponse humorale thymodépendante suite à sa liaison avec son récepteur, le CD40. Il a été démontré par la suite, que le CD154 joue un rôle dans différentes réponses inflammatoires dont certaines peuvent conduire à des processus pathogènes de maladies inflammatoires chroniques et auto-immunes. Depuis quelques années, des études ont montré que les intégrines αIIbβ3, αMβ2, αvβ3 et α5β1 sont de nouveaux récepteurs pour le CD154. Elles jouent un rôle important dans le contrôle de nombreux processus cellulaires tels que la prolifération et l’apoptose cellulaire. Cependant, le rôle biologique de l’interaction du CD154 avec ses récepteurs nouvellement identifiés reste peu étudié. Les cellules T sont des cellules clés de la réponse immunitaire, leur survie et leur persistance constituent unesignature caractéristique de nombreuses maladies inflammatoires et auto-immunes. Lors de ces dernières, les cellules T expriment des niveaux élevés d’intégrines β1. Étant donné le rôle attribué aux intégrines et en particulier aux intégrines β1, dans l'inhibition des événements apoptotiques dans les cellules T, nous étions intéressés d’étudier le rôle de l'interaction CD154 / α5β1 dans la promotion de la survie des cellules T. Dans cette étude, nous avons démontré que le sCD154 est capable de se lier spécifiquement à lʼintégrine α5β1 exprimée par les cellules T, et que cette interaction inhibe l'apoptose cellulaire induite par Fas dans les lignées de cellules T suite à lʼinhibition du clivage de la caspase-8. Nous avons également montré que lʼeffet anti-apoptotique induit par l’axe CD154/α5β1 nʼétait pas restreint au Fas seulement, mais était généralisé aux autres récepteurs de mort de la famille des TNF tel que le TRAIL et le TNF. Plus intéressant encore, nos résultats ont montré que la pré-incubation avec le sCD154 des cellules humaines T CD3+ , protège ces dernières de la mort induite par Fas. Enfin, nous avons montré que comme le sCD154, le mCD154 était capable dʼinhiber la mort induite via les différents récepteurs de mort Fas, TRAIL et TNF, mais seulement par un mécanisme cis-dépendant lorsque le CD154 et lʼintégrine α5β1 sont exprimés à la surface d’une même cellule. Ces résultats soulignent l’importance de l'interaction CD154/α5β1 dans la survie des cellules T et de ce fait, dans leurs rôles biologiques. Cette nouvelle observation inculpe davantage le CD154 et ses récepteurs les intégrines dans l’initiation, mais également dans la progression et la persistance des pathologies inflammatoires et auto-immunes en particulier. L'inhibition spécifique de cet axe peut représenter une cible thérapeutique potentielle dans le traitement des pathologies. / CD154 is a type II glycoprotein belonging to the tumor necrosis factors (TNF). It’s expressed in a transitory way on different cell types. There are two forms of CD154, membrane (mCD154) and soluble (sCD154). Both form are biologically active trimeric structure. CD154 has been identified on its role in the thymus dependent humoral response following its binding with its CD40 receptor. It has subsequently been shown that CD154 plays a role in various inflammatory responses, some of which may lead to pathogenic processes of chronic inflammatory and autoimmune diseases. Recently, studies have shown that other αIIbβ3, αMβ2, αvβ3 and α5β1 receptors belonging to the integrin family could interact with CD154. They play an important role in the control of many cellular processes such as cell proliferation and apoptosis. However, the biological role of the interaction of CD154 with its newly identified receptors remains poorly studied. T lymphocytes are key cells in the immune response. Their survival and persistence is a characteristic signature of numerous inflammatory and autoimmune diseases. In these latter, T cells express high levels of β1 integrins. Given the role attributed to integrins and in particular to β1 integrins in the inhibition of apoptotic events in T cells, we were very interested in studying the role of the CD154 / α5β1 interaction in promoting T cell survival. In this study we have demonstrated that sCD154 is able to specifically bind to T-cell expressed α5β1 integrin. This interaction inhibits Fas-induced apoptosis in Jurkat E6.1 and HUT-78 T cell lines following the inhibition of cleavage of caspase-8. We have also shown that the anti-apoptotic effect induced by the CD154 / α5β1 axis was not restricted to Fas, but was generalized to other TNF-like death receptors such as TRAIL and TNF. More interestingly, our results showed that preincubation with sCD154 of previously activated human T CD3+ cells, protects them from Fas-induced death. Finally, we have shown that, like sCD154, mCD154 was able to inhibit death induced by the different death receptors Fas, TRAIL and TNF, but only by a cis-dependent mechanism when CD154 and α5β1 are expressed at the same time surface of the same cells. These results enhance the importance of the CD154 / α5β1 interaction in the survival of T cells and thus in their biological roles. This new observation further induces CD154 and its integrin receptors in initiation, but also in the progression and persistence of inflammatory and autoimmune pathologies, in particular. The specific inhibition of this axis may represent a potential therapeutic target in the treatment of pathologies.

Integrins have the ability to impact major aspects of epithelial biology including adhesion, migration, invasion, signaling and differentiation, as well as the formation and progression of cancer (Hynes 2002; Srichai and Zent 2010; Anderson et al. 2014). This thesis focuses on how integrins are regulated and function in the context of mammary epithelial biology and breast cancer with a specific focus on the α6 integrin heterodimers (α6β1 and α6β4). These integrins function primarily as receptors for the laminin family of extracellular matrix (ECM) proteins and they have been implicated in mammary gland biology and breast cancer (Friedrichs et al. 1995; Wewer et al. 1997; Mercurio et al. 2001; Margadant and Sonnenberg 2010; Muschler and Streuli 2010; Nistico et al. 2014). The first project investigates how alternative splicing of the α6 subunit impacts the genesis and function of breast cancer stem cells (CSCs). This work revealed that the α6Bβ1 splice variant, but not α6Aβ1, is necessary for the function of breast CSCs because it activates the Hippo transducer TAZ (Zhao et al. 2008a), which is known to be essential for breast CSCs (Cordenonsi et al. 2011). My work also led to the discovery that laminin (LM) 511 is the specific ligand for α6Bβ1 and that autocrine LM511, which is mediated by TAZ, is needed to sustain breast CSCs by functioning as a ‘ECM niche’. An important aspect of this study is the finding that surface-bound LM511 characterizes a small population of cells in human breast tumors with CSC properties. The second project of my thesis concentrated on identifying transcription factors that regulate expression of the β4 subunit. The expression of the α6β4 integrin is repressed during the epithelial-mesenchymal transition (EMT) (Yang et al. 2009) but the contribution of specific transcription factors to this repression is poorly understood. This study revealed that Snai1 is a transcriptional repressor of β4, which is responsible for establishing the PRC2 (Polycomb complex 2)- associated repressive histone mark H3K27Me3. However, I also found that the ability of Snai1 to repress transcription is abrogated by its interaction with Id2. Specifically, I identified the biochemical mechanism for how Id2 regulates Snai1. Id2 binds the SNAG domain of Snai1 that is the docking site for several corepressors (Peinado et al. 2004; Lin et al. 2010b; Dong et al. 2012a). One important consequence of Id2 interacting with Snai1 on the β4 promoter is that it prevents repressive epigenetic modifications. This finding may explain why some epithelial cells express Snai1 and β4 because they also express Id2 (Vincent et al. 2009; Bastea et al. 2012). The repression of the α6β4 integrin during the EMT is consistent with data indicating that this integrin is not expressed in CSCs (Mani et al. 2008; Goel et al. 2012; Goel et al. 2013; Goel et al. 2014). An important question going forward is to understand how the α6β4 integrin contributes to tumor formation. In summary, my thesis provides novel insights into the biology of the α6 integrins that has important implications for the function of these integrins in mammary gland biology and breast cancer, especially our understanding of breast CSCs.

Astrocytes are emerging as important regulators of neural circuit function and behavior in the healthy and diseased nervous system. In a screen for astrocyte molecules that modulate neuronal hyperexcitability we identified multiple components of focal adhesion complexes (FAs) as potent suppressors of genetically- or pharmacologically-induced seizure-like activity. Depletion of astrocytic Tensin, b-integrin, Talin, Focal adhesion kinase (FAK), or matrix metalloproteinase 1 (Mmp1), which degrades extracellular matrix to activate b-integrin receptors, resulted in enhanced recovery from, or resistance to seizure activity. Reciprocally, promoting FA signaling by overexpression of Mmp1 in astrocytes led to enhanced-seizure severity. Blockade of FA signaling in astrocytes led to reduced-astrocytic coverage of the synaptic neuropil and reduced expression of the excitatory amino acid transporter EAAT1. However, upon seizure induction, depletion of FA signaling components resulted in enhanced astrocyte coverage of the synaptic neuropil and a ~2-fold increase in EAAT1 levels compared to controls. Our data indicate that FAs promote astrocyte coverage in neuropil and EAAT1 expression under normal physiological conditions, but in the context of hyperexcitability, FAs negatively regulate the extent of astrocytic processes within neuropil and EAAT1 expression, thereby inhibiting a more rapid recovery from conditions of excessive neuronal activity.

The development and survival of multicellular organisms depends upon the ability of cells to move. Embryogenesis, immune surveillance, wound healing, and metastatic disease are all processes that necessitate effective cellular locomotion. Central to the process of cell motility is the family of integrins, transmembrane cell surface receptors that mediate stable adhesions between cells and their extracellular environment. Many human diseases are associated with aberrant integrin function. Carcinoma cells in particular can hijack integrins, harnessing their mechanical and signaling potential to propagate cell invasion and metastatic disease, one example being integrin α6β4. This integrin, often referred to simply as β4, is defined as an adhesion receptor for the laminin family of extracellular matrix proteins. The role of integrin β4 in potentiating carcinoma invasion is well established, during which it serves both a mechanical and signaling function. miRNAs are short non-coding RNAs that regulate gene expression posttranscriptionally, and data describing the role of extracellular stimuli in governing their expression patterns are sparse. This observation coupled to the increasingly significant role of miRNAs in tumorigenesis prompted us to examine their function as downstream effectors of β4, an integrin closely linked to aggressive disease in breast carcinoma. The work presented in this dissertation documents the first example that integrin expression correlates with specific miRNA patterns. Moreover, integrin β4 status in vitro and in vivo is associated with decreased expression of distinct miRNA families in breast cancer, namely miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, with purported roles in cell motility. Another miRNA, miR-29a, is significantly downregulated in response to de novo expression of β4 in a breast carcinoma cell line, and β4-mediated repression of the miRNA is required for invasion. Another major conclusion of this study is that β4 integrin expression and ligation can regulate the expression of SPARC in breast carcinoma cells. These data reveal distinct mechanisms by which β4 promotes SPARC expression, involving both a miR-29a-mediated process and a TOR-dependent translational mechanism. Our observations establish a link between miRNA expression patterns and cell motility downstream of β4 in the context of breast cancer, and uncover a novel effector of β4-mediated invasion.

Despite the importance of integrins in epithelial cell biology surprisingly little is known about their regulation. It is known that they form hemidesmosomes (HDs), are actively involved in cell contacts during cell migration/invasion, and are key signaling molecules for survival and growth. However, there has been a distinct lack of understanding about what controls the dynamic integrin localization during cell activation and movement. Growth factors, such as EGF, are elevated during wound healing and carcinoma invasion leading to phosphorylation of ITGβ4 and the disassembly of the HD and mobilization of ITGβ4 to actin-rich protrusions. More recently the phosphorylation of a novel site on ITGβ4 (S1424) was found to be distinctly enriched on the trailing edge of migrating cells, suggesting a possible mechanism for the dissociation of ITGβ4 from HDs. Arrestin family member proteins are involved in the regulation of cell surface proteins and vesicular trafficking. In this study, we find that over-expression of arrestin family member ARRDC3 causes internalization and proteosome-dependent degradation of ITGβ4, while decreased levels of ARRDC3 stabilizes ITGβ4 levels. These results lead us to a new mechanism of ITGβ4 internalization, trafficking and degradation. During migration, ARRDC3 co-localizes with ITGβ4 on the lagging edge of cells but has a distinct distribution on the leading edge of cells. Additional immuno co-precipitation experiments demonstrate that ARRDC3 preferentially binds to ITGβ4 when phosphorylated on S1424. Using confocal microscopy, we show that the expression pattern of ARRDC3 on the lagging edge of a migrating cell is identical to the expression pattern of ITGβ4-pS1424. We demonstrate that ARRDC3 expression represses cell proliferation, migration, invasion, growth in soft agar and tumorigenicity. Collectively, our data reveals that ARRDC3 is a negative regulator of β4 integrin and demonstrates how this new pathway impacts biologic processes in stem cell and cancer biology. Additionally, as ARRDC3 is highly expressed in several tissues and conserved across species, our results are likely to be translated to other models.

Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.