Colin K. Choi

Actin and |[alpha]|-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner

Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an α-actinin–actin template that elongates centripetally from nascent adhesions. α-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.

Integrins in cell migration – the actin connection

The connection between integrins and actin is driving the field of cell migration in new directions. Integrins and actin are coupled through a physical linkage, which provides traction for migration. Recent studies show the importance of this linkage in regulating adhesion organization and development. Actin polymerization orchestrates adhesion assembly near the leading edge of a migrating cell, and the dynamic cross-linking of actin filaments promotes adhesion maturation. Breaking the linkage between actin and integrins leads to adhesion disassembly. Recent quantitative studies have revealed points of slippage in the linkage between actin and integrins, showing that it is not always efficient. Regulation of the assembly and organization of adhesions and their linkage to actin relies on signaling pathways that converge on components that control actin polymerization and organization.

Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells

We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front–back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front–back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.

Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro

Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical–basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGF-independent modes of angiogenesis. Like VEGF, sphingosine-1-phosphate also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.

Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions

Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.

Stoichiometry of molecular complexes at adhesions in living cells

We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels, each detecting a different kind of protein. The number and brightness (N&B) analysis, namely, the use of the ratio between the variance and the average intensity to obtain the brightness of molecules, is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin, and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However, at the adhesions, large aggregates leave, forming a hole, during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the correlated fluctuations we determine the composition of the aggregates leaving the adhesions. These aggregates disassemble rapidly in the cytoplasm because large complexes are found only in very close proximity to the adhesions or at their borders.

Fluid shear stress threshold regulates angiogenic sprouting

Significance A great deal of research has investigated the biochemical factors that regulate angiogenic sprouting, but less is known about the role of fluid shear stress. Some studies have suggested distinct regulation by luminal flow within the vessel vs. transmural flow through its walls. In this paper, we demonstrate the existence of a shear stress threshold that when surpassed, induces angiogenic sprouting regardless of whether the shear is applied by primarily luminal or transmural flow. In addition to identifying matrix metalloproteinase 1 as the relevant downstream effector, we use finite-element modeling to predict spatial distributions of shear stress within 3D geometries that experimentally caused localized patterns of sprouting. Together, these studies demonstrate a means by which fluid flow can guide vasculature architecture. The density and architecture of capillary beds that form within a tissue depend on many factors, including local metabolic demand and blood flow. Here, using microfluidic control of local fluid mechanics, we show the existence of a previously unappreciated flow-induced shear stress threshold that triggers angiogenic sprouting. Both intraluminal shear stress over the endothelium and transmural flow through the endothelium above 10 dyn/cm2 triggered endothelial cells to sprout and invade into the underlying matrix, and this threshold is not impacted by the maturation of cell–cell junctions or pressure gradient across the monolayer. Antagonizing VE-cadherin widened cell–cell junctions and reduced the applied shear stress for a given transmural flow rate, but did not affect the shear threshold for sprouting. Furthermore, both transmural and luminal flow induced expression of matrix metalloproteinase 1, and this up-regulation was required for the flow-induced sprouting. Once sprouting was initiated, continuous flow was needed to both sustain sprouting and prevent retraction. To explore the potential ramifications of a shear threshold on the spatial patterning of new sprouts, we used finite-element modeling to predict fluid shear in a variety of geometric settings and then experimentally demonstrated that transmural flow guided preferential sprouting toward paths of draining interstitial fluid flow as might occur to connect capillary beds to venules or lymphatics. In addition, we show that luminal shear increases in local narrowings of vessels to trigger sprouting, perhaps ultimately to normalize shear stress across the vasculature. Together, these studies highlight the role of shear stress in controlling angiogenic sprouting and offer a potential homeostatic mechanism for regulating vascular density.

A DNA-based molecular probe for optically reporting cellular traction forces

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

Engineered materials and the cellular microenvironment: a strengthening interface between cell biology and bioengineering.

Cells constantly probe and respond to a myriad of cues that are present in their local surroundings. The effects of soluble cues are relatively straightforward to manipulate, yet teasing apart how cells transduce signals from the extracellular matrix and neighboring cells has proven to be challenging due to the spatially and mechanically complex adhesive interactions. Over the years, advances in the engineering of biocompatible materials have enabled innovative ways to study adhesion-mediated cell functions, and numerous insights have elucidated the significance of the cellular microenvironment. Here, we highlight some of the major approaches and discuss the potential for future advancement.

Rac1 is deactivated at integrin activation sites through an IQGAP1–filamin-A–RacGAP1 pathway

Summary Cell migration makes a fundamental contribution to both normal physiology and disease pathogenesis. Integrin engagement with extracellular ligands spatially controls, via the cyclical activation and deactivation of the small GTPase Rac1, the dynamic membrane protrusion and cytoskeletal reorganization events that are required for directional migration. Although the pathways that control integrin-mediated Rac1 activation are reasonably well defined, the mechanisms that are responsible for switching off activity are poorly understood. Here, proteomic analysis of activated integrin-associated complexes suggests filamin-A and IQ-motif-containing GTPase-activating protein 1 (IQGAP1) as candidates that link &bgr;1 integrin to Rac1. siRNA-mediated knockdown of either filamin-A or IQGAP1 induced high, dysregulated Rac1 activity during cell spreading on fibronectin. Using immunoprecipitation and immunocytochemistry, filamin-A and IQGAP1 were shown to be part of a complex that is recruited to active &bgr;1 integrin. Mass spectrometric analysis of individual filamin-A, IQGAP1 and Rac1 pull-downs and biochemical analysis, identified RacGAP1 as a novel IQGAP1 binding partner. Further immunoprecipitation and immunocytochemistry analyses demonstrated that RacGAP1 is recruited to IQGAP1 and active &bgr;1 integrin, and that suppression of RacGAP1 expression triggered elevated Rac1 activity during spreading on fibronectin. Consistent with these findings, reduced expression of filamin-A, IQGAP1 or RacGAP1 triggered unconstrained membrane protrusion and disrupted directional cell migration on fibrillar extracellular matrices. These findings suggest a model whereby integrin engagement, followed by filamin-A, IQGAP1 and RacGAP1 recruitment, deactivates Rac1 to constrain its activity spatially and thereby coordinate directional cell migration.