Force mapping during the formation and maturation of cell adhesion sites with multiple optical tweezers

<div><p>Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.</p> </div

Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance

Genetic hardwiring during brain development provides computational architectures for innate neuronal processing. Thus, the paradigmatic chick retinotectal projection, due to its neighborhood preserving, topographic organization, establishes millions of parallel channels for incremental visual field analysis. Retinal axons receive targeting information from quantitative guidance cue gradients. Surprisingly, novel adaptation assays demonstrate that retinal growth cones robustly adapt towards ephrin-A/EphA forward and reverse signals, which provide the major mapping cues. Computational modeling suggests that topographic accuracy and adaptability, though seemingly incompatible, could be reconciled by a novel mechanism of coupled adaptation of signaling channels. Experimentally, we find such ‘co-adaptation’ in retinal growth cones specifically for ephrin-A/EphA signaling. Co-adaptation involves trafficking of unliganded sensors between the surface membrane and recycling endosomes, and is presumably triggered by changes in the lipid composition of membrane microdomains. We propose that co-adaptative desensitization eventually relies on guidance sensor translocation into cis-signaling endosomes to outbalance repulsive trans-signaling

Discreteness of cell–surface contacts affects spatio-temporal dynamics, adhesion, and proliferation of mouse embryonic stem cells

The self-renewal and lineage-specific differentiation of stem cells are regulated by interactions with their microenvironments, called stem cell niche. Stem cells receive both biochemical and biophysical cues from their niche, which leads to the activation of signaling pathways, resulting in the modulation of gene expressions to guide their fate. Most of previous studies are focused on the effect of substrate stiffness using hydrogels with different Young’s moduli, and information is lacking on the effect of the discreteness of cell–substrate contacts on stem cells. Using mouse pluripotent, embryonic stem cells (mESCs) as the model system for early development, we quantitatively investigated the migration, dynamic deformation, and adhesion of mESCs on sparse and dense gelatin nanofibers deposited on glass surfaces, with a continuous layer of gelatin coated on glass substrates as the control. After confirming the maintenance of pluripotency on all the surfaces throughout the experiments, the centroid trajectories were monitored using timelapse imaging. The mean square displacement analysis indicated that both the diffusion coefficient and exponent were largest on sparse nanofibers, while the diffusion coefficient of mESCs on dense nanofibers was comparable to that on the control. Moreover, power spectral analysis of the shape deformation in the Fourier mode indicated that mESCs predominantly underwent elliptic deformation (mode 2), with the largest energy dissipation on sparse nanofibers. These data suggest that mESCs can deform and move on sparse nanofibers owing to the discrete cell–surface contact points. Intriguingly, using a self-developed technique based on laser-induced shock waves, a distinctly larger critical pressure was required to detach cells from nanofibers than from continuous gelatin. This finding suggests that the continuous but weak cell-substrate contacts suppress the deformation-driven mESC migration. As one of the key biological functions of stem cells, the proliferation rate of mESCs on these surfaces was determined. Although the observed difference was not statistically significant, the highest proliferation rate was observed on nanofibers, suggesting that the discreteness of cell–surface contacts can be used to regulate not only spatio-temporal dynamics but also the biological function of pluripotent stem cells

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

Two-Photon 3D Laser Printing Inside Synthetic Cells

Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two-photon 3D laser printing, 2D and 3D hydrogel architectures are manufactured with high precision and nearly arbitrary shape inside preformed giant unilamellar lipid vesicles (GUVs). The required water-soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two-photon printing inside the compartment does not destroy the GUVs. Beyond this proof-of-principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3D printed, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. These experiments show that two-photon 3D laser microprinting can be an important addition to the existing toolbox of synthetic biology

Growth cone response to ephrin gradients produced by microfluidic networks

A microfluidic network (μFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (μCP) step, the protein was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5 were fabricated spanning a total distance of 320 μm. We tested the response of chick retinal ganglion cell (RGC) axons, which are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population, all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5 concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during the detection of a concentration gradient of ephrinA5

Zelluläres Tauziehen: Wie Zellen auf mechanischen Stress antworten

The ability of cells to sense and respond to extracellular forces is critical for cellular and tissue homeostasis. Tension or compression act on our body ubiquitously and cells respond to such mechanical cues by producing intracellular forces on their own. In this article, we briefly highlight the cellular and physical basis driving these phenomena and discuss our recent technical advance to stimulate and monitor the cellular mechanoresponse on a molecular scale

Expressionsstudie von Transport- und Adapterproteinen sowie hormonellen Rezeptoren in der Darmwand von Wildtyp- und NOD2-knockout-Mäusen

In der vorliegenden Arbeit wurde die basale Expression von Transportproteinen, Adapterproteinen und hormonellen Rezeptoren in der Darmwand von WT- und NOD2-KO-Mäusen untersucht. Die Transportproteine, die in dieser Arbeit untersucht wurden, sind DRA, PAT1, NHE3, CFTR und ANO1. Zu den untersuchten Adapterproteinen gehören NHERF1, NHERF2, PDZK1, IKEPP und SNX27. Des Weiteren wurde die Expression der hormonellen Rezeptoren GLP2- und IGF1-Rezeptor bestimmt, welche postoperativ nach einer ausgedehnten Darmresektion eine Adaptation der Darmschleimhaut vermitteln können

Cell Proliferation and Collective Cell Migration During Zebrafish Lateral Line System Development Are Regulated by Ncam/Fgf-Receptor Interactions

The posterior lateral line system (pLLS) of aquatic animals comprises small clustered mechanosensory organs along the side of the animal. They develop from proneuromasts, which are deposited from a migratory primordium on its way to the tip of the tail. We here show, that the Neural Cell Adhesion Molecule Ncam1b is an integral part of the pathways initiating and regulating the development of the pLLS in zebrafish. We find that morpholino-knockdowns of ncam1b (i) reduce cell proliferation within the primordium, (ii) reduce the expression of Fgf target gene erm, (iii) severely affect proneuromast formation, and (iv) affect primordium migration. Ncam1b directly interacts with Fgf receptor Fgfr1a, and a knockdown of fgfr1a causes similar phenotypic changes as observed in ncam1b-morphants. We conclude that Ncam1b is involved in activating proliferation by triggering the expression of erm. In addition, we demonstrate that Ncam1b is required for the expression of chemokine receptor Cxcr7b, which is crucial for directed primordial migration. Finally, we show that the knockdown of ncam1b destabilizes proneuromasts, suggesting a further function of Ncam1b in strengthening the cohesion of proneuromast cells