Cell Shape and Forces in Elastic and Structured Environments: From Single Cells to Organoids

With the advent of mechanobiology, cell shape and forces have emerged as essential elements of cell behavior and fate, in addition to biochemical factors such as growth factors. Cell shape and forces are intrinsically linked to the physical properties of the environment. Extracellular stiffness guides migration of single cells and collectives as well as differentiation and developmental processes. In confined environments, cell division patterns are altered, cell death or extrusion might be initiated, and other modes of cell migration become possible. Tools from materials science such as adhesive micropatterning of soft elastic substrates or direct laser writing of 3D scaffolds have been established to control and quantify cell shape and forces in structured environments. Herein, a review is given on recent experimental and modeling advances in this field, which currently moves from single cells to cell collectives and tissue. A very exciting avenue is the combination of organoids with structured environments, because this will allow one to achieve organotypic function in a controlled setting well suited for long-term and high-throughput culture

Distinct roles of nonmuscle myosin ii isoforms for establishing tension and elasticity during cell morphodynamics

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis

Adaptation of cell spreading to varying fibronectin densities and topographies is facilitated by β1 integrins

Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Several in vitro experiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that β1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with β1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for β1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins

Mechanical stimulation of single cells by reversible host-guest interactions in 3D microscaffolds

Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between β-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors

Adaption der Zellmorphologie durch Aktomyosin-Kontraktilität auf mikrostrukturierten Zellkultursubstraten

Die Zellmorphologie wird durch die räumliche Anordnung des Zytoskeletts und der Aktomyosin-Kontraktilität reguliert. In nicht-muskulären Zellen wird die Kontraktilität von drei verschiedenen Isoformen nicht-muskulärer Myosin II Motorproteine (NM IIA, NM IIB und NM IIC) vermittelt. Die NM II-Isoformen assemblieren dabei zu homotypischen oder heterotypischen Minifilamenten. Anhand des Spannungs-Elastizitätsmodells (TEM) wurde deutlich, dass die Zellmorphologie nicht ausschließlich durch kontraktile Zugspannungen erklärt werden kann, sondern dass auch elastische Komponenten in den Aktinstrukturen erforderlich sind. Bisher ist ungeklärt, wie die verschiedenen NM II-Isoformen den Aufbau der Kontraktilität und die Steuerung der Zellmorphologie vermitteln. Im ersten Teil der Arbeit wurde mit Hilfe von mikrostrukturierten 3D-Substraten gezeigt, dass die Zellmorphologie auch in 3D durch Spannung und Elastizität in den Aktinfasern gesteuert wird und anhand eines erweiterten TEM beschrieben werden kann. Im Hauptteil der Arbeit wurden mittels CRISPR/Cas9 KO-Zelllinien hergestellt, um die Funktionen der einzelnen NM II-Isoformen zu untersuchen. Der Verlust von NM IIA geht mit drastischen Defekten bei der Morphogenese sowie der Bildung von Aktin-Stressfasern einher und hat Auswirkungen auf Zelladhäsion und Zellmigration. Der Knockout von NM IIB und NM IIC zeigt hingegen keine offensichtlichen Effekte auf den Phänotyp. Für eine quantitative Evaluierung der Phänotypen wurden die Zellen auf strukturierten 2D-Substraten analysiert. Für NM IIA und NM IIB zeigten sich klare Effekte, die anhand des TEM erklärt werden können: NM IIA steuert durch den Aufbau von Spannung die Struktur der Aktin-Stressfasern und die Morphogenese der Zellen. NM IIB fungiert als elastisches Element, wodurch die NM IIA-generierten Spannungen ausbalanciert und die Kontraktilität in den Aktin-Stressfasern aufrechterhalten wird. Dadurch zeigte sich, dass NM IIA und NM IIB komplementäre Funktionen beim Aufbau der Aktomyosin-Kontraktilität in nicht-muskulären Zellen ausüben. Für NM IIC konnten keine Funktionen in diesen Assays gezeigt werden. Die komplementären Funktionen von NM IIA und NM IIB wurden in Kolokalisationsstudien weiter analysiert. Die Ergebnisse verdeutlichen, dass sich die Aktomyosin-Kontraktilität nicht wie bisher angenommen, durch stochastische Co-Polymerisation von NM IIA und NM IIB zu einzelnen Minifilamenten gleichermaßen aufbaut, sondern durch präferenzielle Aktivierung von NM IIA-Hexameren initiiert wird. NM IIB co-assembliert hingegen in aktivierte NM IIA-Pionierfilamente, wodurch NM IIA und NM IIB permanent zu heterotypischen Filamenten assemblieren und als eine Einheit fungieren. Zusammenfassend zeigt diese Arbeit, dass NM IIA als Initiator von Minifilamenten und somit der Aktomyosin-Kontraktilität fungiert, wodurch die Bildung von Aktin-Stressfasern und die Morphogenese der Zellen definiert werden. NM IIB nimmt hingegen eine stabilisierende und regulierende Funktion ein, indem es zusätzlich in die Minifilamente eingebaut wird. NM IIB balanciert somit die NM IIA-induzierte Kontraktilität aus und koordiniert dadurch die Dynamik der Aktin-Stressfasern. Die komplementären Funktionen von NM IIA und NM IIB werden dabei durch die direkte Interaktion der Isoformen in heterotypischen Filamenten vermittelt. Auf diese Weise sind Zellen in der Lage, ihre Morphologie zügig und effizient an wechselnde extrazelluläre Gegebenheiten anzupassen

Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions

Integrin-mediated adhesions are convergence points for multiple signaling pathways. Their inner structure and diverse functions can be studied with super-resolution microscopy. Here, we examined the spatial organization within focal adhesions by analyzing several adhesion proteins with structured illumination microscopy (SIM). Paxillin (Pax) serves as a scaffold protein and signaling hub in focal adhesions, and focal adhesion kinase (FAK, also known as PTK2) regulates the dynamics of adhesions. We found that their phosphorylated forms, pPax and pFAK, form spot-like, spatially defined clusters within adhesions in several cell lines and confirmed these findings with additional super-resolution techniques. These clusters showed a more regular separation from each other compared with more randomly distributed signals for FAK or paxillin. Mutational analysis indicated that the active (open) FAK conformation is a prerequisite for the pattern formation of pFAK. Live-cell super-resolution imaging revealed that organization in clusters is preserved over time for FAK constructs; however, distance between clusters is dynamic for FAK, while paxillin is more stable. Combined, these data introduce spatial clusters of pPax and pFAK as substructures in adhesions and highlight the relevance of paxillin-FAK binding for establishing a regular substructure in focal adhesions

Adaptation of cell spreading to varying fibronectin densities and topographies is facilitated by β1 integrins

Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Severalin vitroexperiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that β1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with β1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for β1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins