Erik S. Welf

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Migrating fibroblasts reorient directionality by PI3K-dependent branching and pivoting of protrusions, a mechanism that allows fibroblasts to align with an external chemotactic gradient.

Signaling pathways that control cell migration: models and analysis

Dissecting the intracellular signaling mechanisms that govern the movement of eukaryotic cells presents a major challenge, not only because of the large number of molecular players involved, but even more so because of the dynamic nature of their regulation by both biochemical and mechanical interactions. Computational modeling and analysis have emerged as useful tools for understanding how the physical properties of cells and their microenvironment are coupled with certain biochemical pathways to actuate and control cell motility. In this focused review, we highlight some of the more recent applications of quantitative modeling and analysis in the field of cell migration. Both in modeling and experiment, it has been prudent to follow a reductionist approach in order to characterize what are arguably the principal modules: spatial polarization of signaling pathways, regulation of the actin cytoskeleton, and dynamics of focal adhesions. While it is important that we ‘cut our teeth’ on these subsystems, focusing on the details of certain aspects while ignoring or coarse‐graining others, it is clear that the challenge ahead will be to characterize the couplings between them in an integrated framework. WIREs Syst Biol Med 2011 3 183–190 DOI: 10.1002/wsbm.110.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Stochastic Model of Integrin-Mediated Signaling and Adhesion Dynamics at the Leading Edges of Migrating Cells

Productive cell migration requires the spatiotemporal coordination of cell adhesion, membrane protrusion, and actomyosin-mediated contraction. Integrins, engaged by the extracellular matrix (ECM), nucleate the formation of adhesive contacts at the cells leading edge(s), and maturation of nascent adhesions to form stable focal adhesions constitutes a functional switch between protrusive and contractile activities. To shed additional light on the coupling between integrin-mediated adhesion and membrane protrusion, we have formulated a quantitative model of leading edge dynamics combining mechanistic and phenomenological elements and studied its features through classical bifurcation analysis and stochastic simulation. The model describes in mathematical terms the feedback loops driving, on the one hand, Rac-mediated membrane protrusion and rapid turnover of nascent adhesions, and on the other, myosin-dependent maturation of adhesions that inhibit protrusion at high ECM density. Our results show that the qualitative behavior of the model is most sensitive to parameters characterizing the influence of stable adhesions and myosin. The major predictions of the model, which we subsequently confirmed, are that persistent leading edge protrusion is optimal at an intermediate ECM density, whereas depletion of myosin IIA relieves the repression of protrusion at higher ECM density.

Directional persistence of cell migration coincides with stability of asymmetric intracellular signaling.

It has long been appreciated that spatiotemporal dynamics of cell migration are under the control of intracellular signaling pathways, which are mediated by adhesion receptors and other transducers of extracellular cues. Further, there is ample evidence that aspects of cell migration are stochastic: how else could it exhibit directional persistence over timescales much longer than typical signal transduction processes, punctuated by abrupt changes in direction? Yet the mechanisms by which signaling processes affect those behaviors remain unclear. We have developed analytical methods for relating parallel live-cell microscopy measurements of cell migration dynamics to the intracellular signaling processes that govern them. In this analysis of phosphoinositide 3-kinase signaling in randomly migrating fibroblasts, we observe that hot spots of intense signaling coincide with localized cell protrusion and endure with characteristic lifetimes that correspond to those of cell migration persistence. We further show that distant hot spots are dynamically and stochastically coupled. These results are indicative of a mechanism by which changes in a cells direction of migration are determined by a fragile balance of relatively rapid intracellular signaling processes.

A Spatial Model for Integrin Clustering as a Result of Feedback between Integrin Activation and Integrin Binding

Integrins are transmembrane adhesion receptors that bind extracellular matrix (ECM) proteins and signal bidirectionally to regulate cell adhesion and migration. In many cell types, integrins cluster at cell-ECM contacts to create the foundation for adhesion complexes that transfer force between the cell and the ECM. Even though the temporal and spatial regulation of these integrin clusters is essential for cell migration, how cells regulate their formation is currently unknown. It has been shown that integrin cluster formation is independent of actin stress fiber formation, but requires active (high-affinity) integrins, phosphoinositol-4,5-bisphosphate (PIP2), talin, and immobile ECM ligand. Based on these observations, we propose a minimal model for initial formation of integrin clusters, facilitated by localized activation and binding of integrins to ECM ligands as a result of biochemical feedback between integrin binding and integrin activation. By employing a diffusion-reaction framework for modeling these reactions, we show how spatial organization of bound integrins into clusters may be achieved by a local source of active integrins, namely protein complexes formed on the cytoplasmic tails of bound integrins. Further, we show how such a mechanism can turn small local increases in the concentration of active talin or active integrin into integrin clusters via positive feedback. Our results suggest that the formation of integrin clusters by the proposed mechanism depends on the relationships between production and diffusion of integrin-activating species, and that changes to the relative rates of these processes may affect the resulting properties of integrin clusters.

Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments

The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments.

Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy

The use of propagation invariant Bessel beams has enabled high-resolution subcellular light sheet fluorescence microscopy. However, the energy within the concentric side lobe structure of Bessel beams increases significantly with propagation length, generating unwanted out-of-focus fluorescence that enforces practical limits on the imaging field of view size. Here, we present a light sheet fluorescence microscope that achieves 390 nm isotropic resolution and high optical sectioning strength (i.e., out-of-focus blur is strongly suppressed) over large field of views, without the need for structured illumination or deconvolution-based postprocessing. We demonstrate simultaneous dual-color, high-contrast, and high-dynamic-range time-lapse imaging of migrating cells in complex three-dimensional microenvironments, three-dimensional tracking of clathrin-coated pits, and long-term imaging spanning >10 h and encompassing >2600 time points.

Bidirectional coupling between integrin-mediated signaling and actomyosin mechanics explains matrix-dependent intermittency of leading-edge motility

A physicochemical model is used to describe the coupling of adhesion, cytoskeletal, and signaling dynamics during cell migration. Analysis of stochastic simulations predicts relationships between measurable quantities that reflect partitioning of stress between F-actin–bound adhesions, which act as a molecular clutch, and retrograde F-actin flow.

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Sorting nexin 9 is a multifunctional scaffold protein that coordinates endocytic trafficking, activation of RhoGTPases, and actin nucleation via N-WASP to modulate cancer cell invasion and metastasis.