Biophysical Regulations of Epigenetic State and Notch Signaling in Neural Development Using Microgroove Substrates.

A number of studies have recently shown how surface topography can alter the behavior and differentiation patterns of different types of stem cells. Although the exact mechanisms and molecular pathways involved remain unclear, a consistent portion of the literature points to epigenetic changes induced by nuclear remodeling. In this study, we investigate the behavior of clinically relevant neural populations derived from human pluripotent stem cells when cultured on polydimethylsiloxane microgrooves (3 and 10 µm depth grooves) to investigate what mechanisms are responsible for their differentiation capacity and functional behavior. Our results show that microgrooves enhance cell alignment, modify nuclear geometry, and significantly increase cellular stiffness, which we were able to measure at high resolution with a combination of light and electron microscopy, scanning ion conductance microscopy (SICM), and atomic force microscopy (AFM) coupled with quantitative image analysis. The microgrooves promoted significant changes in the epigenetic landscape, as revealed by the expression of key histone modification markers. The main behavioral change of neural stem cells on microgrooves was an increase of neuronal differentiation under basal conditions on the microgrooves. Through measurements of cleaved Notch1 levels, we found that microgrooves downregulate Notch signaling. We in fact propose that microgroove topography affects the differentiation potential of neural stem cells by indirectly altering Notch signaling through geometric segregation and that this mechanism in parallel with topography-dependent epigenetic modulations acts in concert to enhance stem cell neuronal differentiation.

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

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 micrometers to tens of micrometers. 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.

Force transmission during adhesion-independent migration.

When cells move using integrin-based focal adhesions, they pull in the direction of motion with large, ∼100 Pa, stresses that contract the substrate. Integrin-mediated adhesions, however, are not required for in vivo confined migration. During focal adhesion-free migration, the transmission of propelling forces, and their magnitude and orientation, are not understood. Here, we combine theory and experiments to investigate the forces involved in adhesion-free migration. Using a non-adherent blebbing cell line as a model, we show that actin cortex flows drive cell movement through nonspecific substrate friction. Strikingly, the forces propelling the cell forward are several orders of magnitude lower than during focal-adhesion-based motility. Moreover, the force distribution in adhesion-free migration is inverted: it acts to expand, rather than contract, the substrate in the direction of motion. This fundamentally different mode of force transmission may have implications for cell-cell and cell-substrate interactions during migration in vivo.

Myosin II controls cellular branching morphogenesis and migration in three dimensions by minimizing cell-surface curvature.

In many cases, cell function is intimately linked to cell shape control. We used endothelial cell branching morphogenesis as a model to understand the role of myosin II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell-surface curvature. We find that Rho/ROCK-stimulated myosin II contractility minimizes cell-scale branching by recognizing and minimizing local cell-surface curvature. Using microfabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin II cortical association, where it acts to maintain minimal curvature. The feedback between regulation of myosin II by curvature and control of curvature by myosin II drives cycles of localized cortical myosin II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.

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.

"Stamp-off" to micropattern sparse, multicomponent features.

Spatially patterned subtractive de-inking, a process we term "stamp-off," provides a simple method to generate sparse, multicomponent protein micropatterns. It has been applied to control cell adhesion, study adhesion biology, as well as to micropattern fragile surfaces. This technique can also readily be applied to study nanoscale interactions between cell membrane receptors and surface-immobilized ligands. It is based on conventional microcontact printing and as such requires the same reagents, including photolithographically defined masters, a spin-coater, poly(dimethyl siloxane) (PDMS), and conventional cell culture reagents such as glass coverslips and adhesive proteins. Stamp-off is conceptually simplified into three steps: (1) generation of an appropriate cell culture substrate, PDMS-coated glass, (2) micropatterning with stamp-off, and (3) cell deposition. After elaborating each of these three methods, we discuss limitations of the technique and its applications.

Substrates with engineered step changes in rigidity induce traction force polarity and durotaxis.

Rigidity sensing plays a fundamental role in multiple cell functions ranging from migration, to proliferation and differentiation1-5. During migration, single cells have been reported to preferentially move toward more rigid regions of a substrate in a process termed durotaxis. Durotaxis could contribute to cell migration in wound healing and gastrulation, where local gradients in tissue rigidity have been described. Despite the potential importance of this phenomenon to physiology and disease, it remains unclear how rigidity guides these behaviors and the underlying cellular and molecular mechanisms. To investigate the functional role of subcellular distribution and dynamics of cellular traction forces during durotaxis, we developed a unique microfabrication strategy to generate elastomeric micropost arrays patterned with regions exhibiting two different rigidities juxtaposed next to each other. After initial cell attachment on the rigidity boundary of the micropost array, NIH 3T3 fibroblasts were observed to preferentially migrate toward the rigid region of the micropost array, indicative of durotaxis. Additionally, cells bridging two rigidities across the rigidity boundary on the micropost array developed stronger traction forces on the more rigid side of the substrate indistinguishable from forces generated by cells exclusively seeded on rigid regions of the micropost array. Together, our results highlighted the utility of step-rigidity micropost arrays to investigate the functional role of traction forces in rigidity sensing and durotaxis, suggesting that cells could sense substrate rigidity locally to induce an asymmetrical intracellular traction force distribution to contribute to durotaxis.

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.

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 ß1 but not ß3 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.

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.

Subcellular spatial segregation of integrin subtypes by patterned multicomponent surfaces.

While it is well known that individual integrins are critical mediators of cell behavior, recent work has shown that when multiple types of integrins simultaneously engage the ECM, cell functions are enhanced. However, it is not known how integrins spatially coordinate to regulate cell adhesion because no reliable method exists to segregate integrins on the cell membrane. Here, we use a microcontact printing-based strategy to pattern multiple ECMs that bind distinct integrins in order to study how integrins might interact. In our technique, proteins are first adsorbed uniformly to a poly(dimethyl siloxane) stamp, and then selectively "de-inked." Our strategy overcomes several inherent limitations of conventional microcontact printing, including stamp collapse and limited functionality of the surface patterns. We show that integrins spatially segregate on surfaces patterned with multiple ECMs, as expected. Interestingly, despite spatial segregation of distinct integrins, cells could form adhesions and migrate across multicomponent surfaces as well as they do on single component surfaces. Together, our data indicate that although cells can segregate individual integrins on the cell surface to mediate ECM-specific binding, integrins function cooperatively to guide cell adhesion and migration.

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.

Micropatterned dynamically adhesive substrates for cell migration.

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.

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.

Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis.

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.

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.

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.

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.

Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct.

We have previously demonstrated that vasopressin increases the water permeability of the inner medullary collecting duct (IMCD) by inducing trafficking of aquaporin-2 to the apical plasma membrane and that this response is dependent on intracellular calcium mobilization and calmodulin activation. Here, we address the hypothesis that this water permeability response is mediated in part through activation of the calcium/calmodulin-dependent myosin light chain kinase (MLCK) and regulation of non-muscle myosin II. Immunoblotting and immunocytochemistry demonstrated the presence of MLCK, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32P incubation of the inner medulla followed by autoradiography of two-dimensional gels demonstrated increased 32P labeling of both isoforms in response to the V2 receptor agonist [deamino-Cys1,D-Arg8]vasopressin (DDAVP). Time course studies of MLC phosphorylation in IMCD suspensions (using immunoblotting with anti-phospho-MLC antibodies) showed that the increase in phosphorylation could be detected as early as 30 s after exposure to vasopressin. The MLCK inhibitor ML-7 blocked the DDAVP-induced MLC phosphorylation and substantially reduced [Arg8]vasopressin (AVP)-stimulated water permeability. AVP-induced MLC phosphorylation was associated with a rearrangement of actin filaments (Alexa Fluor 568-phalloidin) in primary cultures of IMCD cells. These results demonstrate that MLC phosphorylation by MLCK represents a downstream effect of AVP-activated calcium/calmodulin signaling in IMCD cells and point to a role for non-muscle myosin II in regulation of water permeability by vasopressin.