Ravi A. Desai

Mechanical regulation of cell function with geometrically modulated elastomeric substrates

We report the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility predicted later stem cell fate decisions in single cells.

Nanotechnology for Cell–Substrate Interactions

In the pursuit to understand the interaction between cells and their underlying substrates, the life sciences are beginning to incorporate micro- and nanotechnology-based tools to probe and measure cells. The development of these tools portends endless possibilities for new insights into the fundamental relationships between cells and their surrounding microenvironment that underlie the physiology of human tissue. Here, we review techniques and tools that have been used to study how a cell responds to the physical factors in its environment. We also discuss unanswered questions that could be addressed by these approaches to better elucidate the molecular processes and mechanical forces that dominate the interactions between cells and their physical scaffolds.

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8+ T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.

Cell polarity triggered by cell-cell adhesion via E-cadherin

Cell polarity is orchestrated by numerous extracellular cues, and guides events such as chemotaxis, mitosis and wound healing. In scrape-wound assays of cell monolayers, wound-edge cells orient their centrosomes towards the wound, a process that appears to depend on the formation of new cell–extracellular-matrix adhesions as cells spread into the wound. In direct contrast to scrape-wounded cells, isolated cells without cell-cell contacts failed to polarize, suggesting that asymmetry of cell-cell adhesions resulting from monolayer disruption might contribute to polarization. By using micropatterned substrates to engineer such asymmetries in kidney epithelial cells, we found that cell-cell contact induced displacement of the nucleus towards the contact, and also caused centrosomal reorientation and lamellipodial ruffling to the distal side of the nucleus. Upon release from micropatterned constraints, cells exhibited directed migration away from the cell-cell contact. Disrupting E-cadherin engagement randomized nuclear position and lamellipodial ruffling in patterned cultures, and abrogated scrape-wound-induced cell reorientation, but not migration rate. Polarity that was induced by cell-cell contact required an intact actin cytoskeleton and Cdc42 activity, but not RhoA or Rac signaling. Together, these findings demonstrate a novel role for cell-cell adhesion in polarization, and have implications for wound healing and developmental patterning.

Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity

We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. This protocol describes how to fabricate the silicon micropost array masters (∼2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1–14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation.

Cell mechanics control rapid transitions between blebs and lamellipodia during migration

Protrusion formation is an essential step during cell migration. Cells migrating in three-dimensional environments and in vivo can form a wide variety of protrusion types, including actin polymerization-driven lamellipodia, and contractility-driven blebs. The ability to switch between different protrusions has been proposed to facilitate motility in complex environments and to promote cancer dissemination. However, plasticity in protrusion formation has so far mostly been investigated in the context of transitions between amoeboid and mesenchymal migration modes, which involve substantial changes in overall cell morphology. As a result, the minimal requirements of transitions between blebs and lamellipodia, as well as the time scales on which they occur, remain unknown. To address these questions, we investigated protrusion switching during cell migration at the single cell level. Using cells that can be induced to form either blebs or lamellipodia, we systematically assessed the mechanical requirements, as well as the dynamics, of switching between protrusion types. We demonstrate that shifting the balance between actin protrusivity and actomyosin contractility leads to immediate transitions between blebs and lamellipodia in migrating cells. Switching occurred without changes in global cell shape, polarity, or cell adhesion. Furthermore, rapid transitions between blebs and lamellipodia could also be triggered upon changes in substrate adhesion during migration on micropatterned surfaces. Together, our data reveal that the type of protrusion formed by migrating cells can be dynamically controlled independently of overall cell morphology, suggesting that protrusion formation is an autonomous module in the regulatory network that controls the plasticity of cell migration.

Activation of beta 1 but not beta 3 integrin increases cell traction forces

Cell‐generated traction forces induce integrin activation, leading to focal adhesion growth and cell spreading. It remains unknown, however, whether integrin activation feeds back to impact the generation of cytoskeletal tension. Here, we used elastomeric micropost arrays to measure cellular traction forces in wildtype and integrin‐null cells. We report that activation of β1 but not β3 integrin, by either increasing density of immobilized fibronectin or treating with manganese, elicited fibroblast spreading and cytoskeletal tension. Furthermore, this force generation required Rho kinase and myosin activity. These findings suggest that integrin activation and cell traction forces comprise a bi‐directional signaling unit of cell adhesion.

Contact inhibition of locomotion probabilities drive solitary versus collective cell migration.

Contact inhibition of locomotion (CIL) is the process whereby cells collide, cease migrating in the direction of the collision, and repolarize their migration machinery away from the collision. Quantitative analysis of CIL has remained elusive because cell-to-cell collisions are infrequent in traditional cell culture. Moreover, whereas CIL predicts mutual cell repulsion and ‘scattering’ of cells, the same cells in vivo are observed to undergo CIL at some developmental times and collective cell migration at others. It remains unclear whether CIL is simply absent during collective cell migration, or if the two processes coexist and are perhaps even related. Here, we used micropatterned stripes of extracellular matrix to restrict cell migration to linear paths such that cells polarized in one of two directions and collisions between cells occurred frequently and consistently, permitting quantitative and unbiased analysis of CIL. Observing repolarization events in different contexts, including head-to-head collision, head-to-tail collision, collision with an inert barrier, or no collision, and describing polarization as a two-state transition indicated that CIL occurs probabilistically, and most strongly upon head-to-head collisions. In addition to strong CIL, we also observed ‘trains’ of cells moving collectively with high persistence that appeared to emerge from single cells. To reconcile these seemingly conflicting observations of CIL and collective cell migration, we constructed an agent-based model to simulate our experiments. Our model quantitatively predicted the emergence of collective migration, and demonstrated the sensitivity of such emergence to the probability of CIL. Thus CIL and collective migration can coexist, and in fact a shift in CIL probabilities may underlie transitions between solitary cell migration and collective cell migration. Taken together, our data demonstrate the emergence of persistently polarized, collective cell movement arising from CIL between colliding cells.

Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning

Summary During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from microns to tens of microns. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes.

Micropatterned Multicolor Dynamically Adhesive Substrates to Control Cell Adhesion and Multicellular Organization

We present a novel technique to examine cell–cell interactions and directed cell migration using micropatterned substrates of three distinct regions: an adhesive region, a nonadhesive region, and a dynamically adhesive region switched by addition of a soluble factor to the medium. Combining microcontact printing with avidin–biotin capture chemistry, we pattern nonadhesive regions of avidin that become adhesive through the capture of biotinylated fibronectin. Our strategy overcomes several limitations of current two-color dynamically adhesive substrates by incorporating a third, permanently nonadhesive region. Having three spatially and functionally distinct regions allows for the realization of more complex configurations of cellular cocultures as well as intricate interface geometries between two cell populations for diverse heterotypic cell–cell interaction studies. We can now achieve spatial control over the path and direction of migration in addition to temporal control of the onset of migration, enabling studies that better recapitulate coordinated multicellular migration and organization in vitro. We confirm that cellular behavior is unaltered on captured biotinylated fibronectin as compared to printed fibronectin by examining the cells’ ability to spread, form adhesions, and migrate. We demonstrate the versatility of this approach in studies of migration and cellular cocultures, and further highlight its utility by probing Notch–Delta juxtacrine signaling at a patterned interface.