Bernhard Wehrle-Haller

Cell behaviour on micropatterned substrata: limits of extracellular matrix geometry for spreading and adhesion

Cell adhesion, spreading and migration require the dynamic formation and dispersal of contacts with the extracellular matrix (ECM). In vivo, the number, availability and distribution of ECM binding sites dictate the shape of a cell and determine its mobility. To analyse the geometrical limits of ECM binding sites required for cell attachment and spreading, we used microcontact printing to produce regular patterns of ECM protein dots of defined size separated by nonadhesive regions. Cells cultured on these substrata adhere to and spread on ECM regions as small as 0.1 μm2, when spacing between dots is less than 5 μm. Spacing of 5-25 μm induces a cell to adapt its shape to the ECM pattern. The ability to spread and migrate on dots ≥1 μm2 ceases when the dot separation is ≥30 μm. The extent of cell spreading is directly correlated to the total substratum coverage with ECM-proteins, but irrespective of the geometrical pattern. An optimal spreading extent is reached at a surface coating above 15%. Knowledge of these geometrical limits is essential for an understanding of cell adhesion and migration, and for the design of artificial surfaces that optimally interact with cells in a living tissue.

Marching at the front and dragging behind: differential αVβ3-integrin turnover regulates focal adhesion behavior

Integrins are cell–substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed αVβ3-integrin dynamics in migrating cells using a green fluorescent protein–tagged β3-integrin chain. At the cell front, adhesion sites containing αVβ3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of β3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast β3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin–dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

Actin, microtubules and focal adhesion dynamics during cell migration

Cell migration is a complex cellular behavior that results from the coordinated changes in the actin cytoskeleton and the controlled formation and dispersal of cell-substrate adhesion sites. While the actin cytoskeleton provides the driving force at the cell front, the microtubule network assumes a regulatory function in coordinating rear retraction. The polarity within migrating cells is further highlighted by the stationary behavior of focal adhesions in the front and their sliding in trailing ends. We discuss here the cross-talk of the actin cytoskeleton with the microtubule network and the potential mechanisms that control the differential behavior of focal adhesions sites during cell migration.

Multiscale Modeling of Form and Function

Topobiology posits that morphogenesis is driven by differential adhesive interactions among heterogeneous cell populations. This paradigm has been revised to include force-dependent molecular switches, cell and tissue tension, and reciprocal interactions with the microenvironment. It is now appreciated that tissue development is executed through conserved decision-making modules that operate on multiple length scales from the molecular and subcellular level through to the cell and tissue level and that these regulatory mechanisms specify cell and tissue fate by modifying the context of cellular signaling and gene expression. Here, we discuss the origin of these decision-making modules and illustrate how emergent properties of adhesion-directed multicellular structures sculpt the tissue, promote its functionality, and maintain its homeostasis through spatial segregation and organization of anchored proteins and secreted factors and through emergent properties of tissues, including tension fields and energy optimization.

Assembly and disassembly of cell matrix adhesions.

The formation of tissues and organs requires cells to adhere to each other and/or to migrate and polarize in contact with components of the extracellular matrix. The connection between the cytoskeleton and the extracellular environment is provided by heterodimeric transmembrane receptors of the integrin family. In response to extracellular ligand binding, integrins undergo a conformational switch that permits the recruitment of cytoplasmic adapter proteins, eventually linking the integrin receptors to the actin cytoskeleton, progressively forming highly complex cell-matrix adhesions. A major challenge in the field consists in identifying the regulatory mechanisms, which drive the assembly of cell-matrix adhesions as they are based on posttranslational modifications as well as allosteric conformational changes caused by protein-protein as well as protein-lipid interactions. In response to mechanical tension, generated either by intra-cellular acto-myosin contraction, shear stress or mechanical strain on the extracellular scaffold, the composition and signaling of cell-matrix adhesion changes, leading either to increased anchorage or controlled disassembly of cell matrix adhesions, both processes critically involved in cell migration. The aim of this review is to provide insight into the mechanisms leading to the progressive assembly of focal adhesions, how they are modulated in response to mechanical challenges and which mechanisms are used for their disassembly.

Structure and function of focal adhesions

Integrin-dependent cell adhesions come in different shapes and serve in different cell types for tasks ranging from cell-adhesion, migration, and the remodeling of the extracellular matrix to the formation and stabilization of immunological and chemical synapses. A major challenge consists in the identification of adhesion-specific as well as common regulatory mechanisms, motivating the need for a deeper analysis of protein-protein interactions in the context of intact focal adhesions. Specifically, it is critical to understand how small differences in binding of integrins to extracellular ligands and/or cytoplasmic adapter proteins affect the assembly and function of an entire focal adhesion. By using the talin-integrin pair as a starting point, I would like to discuss how specific protein-protein and protein-lipid interactions can control the behavior and function of focal adhesions. By responding to chemical and mechanical cues several allosterically regulated proteins create a dynamic multifunctional protein network that provides both adhesion to the extracellular matrix as well as intracellular signaling in response to mechanical changes in the cellular environment.

New PI(4,5)P2- and membrane proximal integrin–binding motifs in the talin head control β3-integrin clustering

A talin intermolecular interaction autoinhibits its own activation and regulates β3-integrin binding. When bound, β3-integrin undergoes structural alterations that prevent its β and α subunits from associating, maintaining β3-integrins clustering capability.

Integrin-dependent pathologies

Cell adhesion and migration are essential for embryonic development, tissue regeneration, and immune defence. The physical link between the extracellular substrate and the actin cytoskeleton is mediated by receptors of the integrin family and a large set of adaptor proteins. During cell migration this physical link is dynamically modified, allowing the cell to sense and adapt to the microenvironment. This includes the formation of integrin clusters at the cell front, their stabilization in the cell body and subsequent disassembly of these clusters at the rear of the cell. The modulation of the adhesion strength of the cell to the substrate is regulated by the affinity switch of integrin molecules and increased avidity through clustering of integrins. Here we explain how integrins mediate cell migration and how genetic defects of integrins and their adaptors lead to cellular dysfunction and generate pathological situations. Copyright

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases.

An adhesion molecule in free-living Dictyostelium amoebae with integrin beta features

The study of free‐living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin β chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well‐characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin β chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.