Receptor clustering control and associated force sensing by surface patterning: when force matters
2015; Future Medicine; Volume: 10; Issue: 5 Linguagem: Inglês
10.2217/nnm.14.234
ISSN1748-6963
AutoresElisabetta Ada Cavalcanti‐Adam, Joachim P. Spatz,
Tópico(s)Cell Adhesion Molecules Research
ResumoNanomedicineVol. 10, No. 5 EditorialFree AccessReceptor clustering control and associated force sensing by surface patterning: when force mattersElisabetta Ada Cavalcanti-Adam & Joachim P SpatzElisabetta Ada Cavalcanti-AdamMax Planck Institute for Intelligent Systems, Department of New Materials & Biosystems, Heisenbergstr. 3, D-70569 Stuttgart, GermanyUniversity of Heidelberg, Department of Biophysical Chemistry, INF 253, D-69120 Heidelberg, Germany & Joachim P SpatzAuthor for correspondence: E-mail Address: spatz@is.mpg.deMax Planck Institute for Intelligent Systems, Department of New Materials & Biosystems, Heisenbergstr. 3, D-70569 Stuttgart, GermanyUniversity of Heidelberg, Department of Biophysical Chemistry, INF 253, D-69120 Heidelberg, GermanyPublished Online:30 Mar 2015https://doi.org/10.2217/nnm.14.234AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: biointerfacesextracellular matrixintegrinsnanopatternsreceptor clusteringtension sensorNanostructured and chemically functionalized materials which mimic architectural and mechanical features of natural cell microenvironments hold promise for a better understanding and control of cell physiological processes through molecular and nanoscale interactions. Ultimately, the design of defined scaffolds for tissue engineering based on these material properties will advance regenerative medicine. The clustering of transmembrane receptors into defined nanoscale structures triggers and regulates specific signaling networks with unprecedented precision and is involved in transducing forces between the cell and the matrix. Only few material-based technologies exist today that enable the local control of receptor clustering and are able to measure mechanotransduction-based cellular reactions. Receptor clustering and regulatory ligand–receptor interactions stimulate a variety of biological processes. Herein lies an opportunity for interdisciplinary efforts between the fields of engineering, chemistry and biology to design new materials with the aim of controlling and quantifying nanoscale and molecular interactions at cellular boundaries. These efforts, inspired by the cell microenvironment, have recently led to the creation of nanostructured surfaces for controlling and guiding cell adhesion and function in a predictable manner [1,2].The cell microenvironment contains chemical and physical cues that arise from a complex, albeit defined, architecture of extracellular matrix networks. Achieving a defined spatial patterning of extracellular matrix cues at the nanoscale, while independently tuning the chemical and physical properties of surfaces, has been a challenge. Although the achievements made thus far have created a more detailed picture of how cells interact with their microenvironment, it has also raised new questions.Current techniques used to investigate the effects of extracellular matrix ligand presentation on cell functions work by independently manipulating variables like ligand density, clustering and spacing [3–5]. The application of surfaces that present a specific spatial pattern of molecules and peptides at the nanoscale have elucidated the minimal requirements needed for activating signaling networks. In particular, strong interest has been devoted to understanding the spatial aspects of focal adhesion maturation, the assembly of discrete structures upon integrin binding to the extracellular matrix and large-scale clustering of hundreds of receptors. Focal adhesions transmit tensile stresses from the extracellular space to the cytoskeleton, thereby converting force cues into biochemical signals that regulate cell functions [6].Molecular nanopatterns for controlling transmembrane clusteringNanoscale clustering of integrin ligands promotes substrate attachment and focal adhesion reinforcement. The maximum distance between integrin-binding sequences in fibronectin that will allow attachment has been estimated to be approximately 60 nm [7]. The corresponding spacing of integrin binding sites on the cytoplasmic antiparallel talin dimer is approximately 50 nm [8]. This suggests that the nanoscale ligand arrangement of the extracellular matrix functions as a template for guiding the assembly of the adhesion complex inside the cell. Accordingly, when the spacing of patterned ligands on a surface exceeds the size of the complex, unstable focal adhesions are formed.First attempts to locally control integrin receptor activation and clustering were reported using self-assembled PEG-based monolayers on gold or glass interfaces. The PEG hydrogels were made of linear and star-shaped macromolecules with oligo ethylene glycol end groups [9,10]. During production hydrogels were functionalized with the peptide motif Arg-Gly-Asp (RGD), an integrin-binding fibronectin fragment. These hydrogels remarkably improved the ability to study the interaction of integrins with RGD-peptides immobilized on surfaces, because ethylene glycol reduces cell–surface interactions to a minimum. Concerning the density of the bound RGD domains, a minimum average spacing between individual RGD peptides of more than 400 nm could be achieved. However, this method was unsuccessful in achieving spatial control over RGD peptide density at the length scale of single receptor clusters and creating ligand arrangements with specific patterns. The lack of spatially controlled chemical templates with nanometer resolution resulted in spatially inhomogeneous ligand distribution.Diblock copolymers form well-ordered structures that are defined by the macromolecular characteristics both in bulk and at surfaces [4]. Their dimensions and geometries fit molecular length scales of molecular complexes in cells that can steer signaling networks (between 10 and 300 nm). Block copolymer micelle nanolithography enables the well-ordered and stable deposition of subnanometer large gold nanoparticles onto PEG hydrogels or PEG-passivated glass substrates [11]. Each gold nanoparticle can be covalently functionalized with peptides, antibodies or cytokines, turning it into a 'chemical hand'. Based on its specific size, it can grab exactly one transmembrane receptor. The way the gold nanoparticles are distributed on the surface therefore determines the relative position of the adhering cellular transmembrane receptors. This technology has underscored the importance of local molecular order and spacing gradients for adhesion processes. Accordingly, the critical clustering length scale for integrins was determined at 58 nm for focal adhesion formation [4,12]. Lately, a variety of self-assembled and biofunctionalized polymer domains have now been used to successfully regulate signaling networks [13]. In future, the design of potent multivalent conjugates that can organize cell receptors into nanoscale clusters and control cell fate will further impact this research field [1,14,15].Whereas knowledge on nanoscale ligand spacing has clarified the importance of receptor binding and clustering, a fundamental question remains: which factors determine ligand spacing and how do they influence cell mechanics, development and eventually physiology of cells?Nanoscale regulation of forces at the cell–material interfaceThe clustering of transmembrane receptors has been linked to the maturation of signaling networks. It is also often associated with various forces, which act on the associated protein complex. Reinforcement and increased stability of focal adhesions are typically observed on substrates with periodic patterns of nanostructures. One explanation for this may be the cellular forces exerted through the actin cytoskeleton. This assumption is, thus far, supported only by physicochemical models that show the level of stress at the cytoskeleton to be one determining factor for driving the transition of cellular adhesions from a stable state (at high ligand densities, i.e., ligand spacing 58 nm) [16]. The model assumes that there is no direct influence of ligand density on adhesion stability.However, only through recent advances in molecular tension-based fluorescence microscopy techniques, has the range of force amplitudes that can be sensed and transduced by single integrins been revealed [17,18]. In addition, it was observed that the bond formed between integrins and their ligands, specifically an RGD anchored to the surface using biotin–streptavidin linkage, is so strong that the receptors detached from the ligand [19]. A possible explanation for this is that several integrins cluster at adhesion sites and cooperate in generating high forces. The combined use of nanopatterned surfaces with molecular tension probes has allowed simultaneous control over receptor clustering with nanometer precision and has made it possible to measure forces at single receptor binding sites with pN resolution [20]. With these techniques it was experimentally proven for the first time that the mechanism of sensing ligand spacing is indeed force mediated. In fact, it was shown that the transmission of myosin-generated tension to individual receptors only took place inside cells that were adhering to nanopatterned surfaces with ligands spaced apart less than 58 nm. Early during the adhesion process, the force per single integrin is independent of ligand spacing and, therefore, of lateral clustering of receptors. However, ligands must be spaced less than 58 nm apart for tensile forces to increase to more than 3 pN. Although the total tension generated by the cell increases as the adhesion site maturates, the tension per integrin receptor is continuously maintained at a constant value of 6 pN. These data suggest that a growing number of surface-bound integrins, rather than an increase in the binding force of single receptor-ligand sites, is responsible for the exertion of continuously increasing traction forces on the surface. The concept that optimal ligand spacing allows surface-bound integrins to counteract the forces generated by the actomyosin cytoskeleton by increasing their average tension and forming stable focal adhesion, complements the idea that talin and actin-generated forces are responsible for controlling focal adhesion dynamics. Additionally, it highlights the importance of the control of receptor clustering at the interface between cells and materials.Conclusion & outlookNanostructured materials are ideally suited to study the impact that spatial properties at the single molecule level have on cell adhesion. These materials in combination with molecular tension-based fluorescence microscopy make it possible to explore the mechanical response of cells as they sense the nanoscale organization of their environment. In future, these techniques will help to clarify how the relationship between cell receptor clustering and forces impacts cell signaling and functions. For example, it will become possible to determine the mechanisms used by cells to pull and tug on their environment. This could bring forth new insights into physiological and pathological processes, such as wound healing and cancer. In the field of tissue engineering and regenerative application, nanostructured materials might also be used – in addition to the static control of cell binding – for the dynamic guidance of cell forces that are necessary for the creation of stable adhesions at the material interface.Financial & competing interests disclosureThis work was supported by the Max Planck Society. JP Spatz is the Weston Visiting Professor at the Weizmann Institute of Science and is a member of the Heidelberg cluster of excellence CellNetworks. EA Cavalcanti-Adam is member of the SFB TRR79 at the University of Heidelberg. EA Cavalcanti-Adam and JP Spatz are members of the SFB 1129 at the University of Heidelberg. 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JP Spatz is the Weston Visiting Professor at the Weizmann Institute of Science and is a member of the Heidelberg cluster of excellence CellNetworks. EA Cavalcanti-Adam is member of the SFB TRR79 at the University of Heidelberg. EA Cavalcanti-Adam and JP Spatz are members of the SFB 1129 at the University of Heidelberg. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download
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