Benjamin Lin

A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues

Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.

Diverse Sensitivity Thresholds in Dynamic Signaling Responses by Social Amoebae

Dictyostelium cells with increased responsiveness to chemoattractant may promote propagation of the initial signal across the cell population. Genetically Identical, But Not Equally Sensitive Individual cells of the social amoeba Dictyostelium can organize to form multicellular ensembles, a process in which the chemoattractant cAMP (cyclic adenosine monophosphate) triggers production of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to induce polarization of the chemotaxing cells. Cell population responses have generally been averaged; however, Wang et al. found that not all cells in a group of genetically identical Dictyostelium cells produced PIP3 in response to cAMP and that more cells produced PIP3 as the cAMP concentration increased. Furthermore, modeling and experiments suggested that an amplification step occurred downstream of an adaptive signaling network and that different cells had different amplification thresholds. Different sensitivities and thresholds for signal amplification may enable highly responsive individual cells to promote chemotaxis of an entire population of Dictyostelium cells and, thus, mediate collective behavior. The complex transition from a single-cell to a multicellular life form during the formation of a fruiting body by the amoeba Dictyostelium discoideum is accompanied by a pulsatile collective signaling process that instigates chemotaxis of the constituent cells. Although the cells used for the analysis of this phenomenon are normally genetically identical (isogenic), it is not clear whether they are equally responsive to the waves of the signaling stimulus, nor is it clear how responses across the population influence collective cell behavior. Here, we found that isogenic Dictyostelium cells displayed differing sensitivities to the chemoattractant cyclic adenosine monophosphate (cAMP). Furthermore, the resulting signaling responses could be explained by a model in which cells are refractory to further stimulation for 5 to 6 min after the initial input and the signaling output is amplified, with the amplification threshold varying across the cells in the population. This pathway structure could explain intracellular amplification of the chemoattractant gradient during cell migration. The new model predicts that diverse cell responsiveness can facilitate collective cell behavior, specifically due to the presence of a small number of cells in the population with increased responsiveness that aid in propagating the initial cAMP signaling wave across the cell population.

Synthetic spatially graded Rac activation drives cell polarization and movement

Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.

Pre‐Exposure of Human Adipose Mesenchymal Stem Cells to Soluble Factors Enhances Their Homing to Brain Cancer

Recent research advances have established mesenchymal stem cells (MSCs) as a promising vehicle for therapeutic delivery. Their intrinsic tropism for brain injury and brain tumors, their lack of immunogenicity, and their ability to breach the blood‐brain barrier make these cells an attractive potential treatment of brain disorders, including brain cancer. Despite these advantages, the efficiency of MSC homing to the brain has been limited in commonly used protocols, hindering the feasibility of such therapies. In the present study, we report a reproducible, comprehensive, cell culture‐based approach to enhance human adipose‐derived MSC (hAMSC) engraftment to brain tumors. We used micro‐ and nanotechnological tools to systematically model several steps in the putative homing process. By pre‐exposing hAMSCs to glioma‐conditioned media and the extracellular matrix proteins fibronectin and laminin, we achieved significant enhancements of the individual homing steps in vitro. This homing was confirmed in an in vivo rodent model of brain cancer. This comprehensive, cell‐conditioning approach provides a novel method to enhance stem cell homing to gliomas and, potentially, other neurological disorders.

Interplay between chemotaxis and contact inhibition of locomotion determines exploratory cell migration

Directed cell migration in native environments is influenced by multiple migratory cues. These cues may include simultaneously occurring attractive soluble growth factor gradients and repulsive effects arising from cell-cell contact, termed contact inhibition of locomotion (CIL). How single cells reconcile potentially conflicting cues remains poorly understood. Here we show that a dynamic crosstalk between epidermal growth factor (EGF) mediated chemotaxis and CIL guide metastatic breast cancer cell motility, whereby cells become progressively insensitive to CIL in a chemotactic input-dependent manner. This balance is determined via integration of protrusion-enhancing signaling from EGF gradients and protrusion-suppressing signaling induced by CIL, mediated in part through EphB. Our results further suggest that EphB and EGF signaling inputs control protrusion formation by converging onto regulation of phosphatidylinositol 3-kinase (PI3K). We propose that this intricate interplay may enhance the spread of loose cell ensembles in pathophysiological conditions such as cancer, and possibly other physiological settings.

Modelling cell polarization driven by synthetic spatially graded Rac activation.

The small GTPase Rac is known to be an important regulator of cell polarization, cytoskeletal reorganization, and motility of mammalian cells. In recent microfluidic experiments, HeLa cells endowed with appropriate constructs were subjected to gradients of the small molecule rapamycin leading to synthetic membrane recruitment of a Rac activator and direct graded activation of membrane-associated Rac. Rac activation could thus be triggered independent of upstream signaling mechanisms otherwise responsible for transducing activating gradient signals. The response of the cells to such stimulation depended on exceeding a threshold of activated Rac. Here we develop a minimal reaction-diffusion model for the GTPase network alone and for GTPase-phosphoinositide crosstalk that is consistent with experimental observations for the polarization of the cells. The modeling suggests that mutual inhibition is a more likely mode of cell polarization than positive feedback of Rac onto its own activation. We use a new analytical tool, Local Perturbation Analysis, to approximate the partial differential equations by ordinary differential equations for local and global variables. This method helps to analyze the parameter space and behaviour of the proposed models. The models and experiments suggest that (1) spatially uniform stimulation serves to sensitize a cell to applied gradients. (2) Feedback between phosphoinositides and Rho GTPases sensitizes a cell. (3) Cell lengthening/flattening accompanying polarization can increase the sensitivity of a cell and stabilize an otherwise unstable polarization.

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

Actin filament formation plays an essential role in cell movement, and posttranslational modifications regulate actin filament assembly. Prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actins in HeLa cells and catalyzes prolyl hydroxylation of actin. Inhibition of PHD3 activity or expression increases F-actin formation and cell motility.

Toward total synthesis of cell function: Reconstituting cell dynamics with synthetic biology

Synthetic biology approaches have revealed new insights into cellular proliferation, differentiation, phagocytosis, and chemotaxis. Gloss Synthetic biology approaches have contributed to our understanding of many cellular processes. In this Review, which contains 4 figures and 38 references, we show how synthetic biology approaches have revealed new insights into cellular proliferation, cellular differentiation, phagocytosis, and chemotaxis. The focus of this Review will be on case studies that highlight the applications of synthetic biology in biological experiments. Biological phenomena, such as cellular differentiation and phagocytosis, are fundamental processes that enable cells to fulfill important physiological roles in multicellular organisms. In the field of synthetic biology, the study of these behaviors relies on the use of a broad range of molecular tools that enable the real-time manipulation and measurement of key components in the underlying signaling pathways. This Review will focus on a subset of synthetic biology tools known as bottom-up techniques, which use technologies such as optogenetics and chemically induced dimerization to reconstitute cellular behavior in cells. These techniques have been crucial not only in revealing causal relationships within signaling networks but also in identifying the minimal signaling components that are necessary for a given cellular function. We discuss studies that used these systems in a broad range of cellular and molecular phenomena, including the time-dependent modulation of protein activity in cellular proliferation and differentiation, the reconstitution of phagocytosis, the reconstitution of chemotaxis, and the regulation of actin reorganization. Finally, we discuss the potential contribution of synthetic biology to medicine.

Spatial Manipulation with Microfluidics

Biochemical gradients convey information through space, time, and concentration, and are ultimately capable of spatially resolving distinct cellular phenotypes, such as differentiation, proliferation, and migration. How these gradients develop, evolve, and function during development, homeostasis, and various disease states is a subject of intense interest across a variety of disciplines. Microfluidic technologies have become essential tools for investigating gradient sensing in vitro due to their ability to precisely manipulate fluids on demand in well-controlled environments at cellular length scales. This review will highlight their utility for studying gradient sensing along with relevant applications to biology.

Visualizing Molecular Diffusion through Passive Permeability Barriers in Cells: Conventional and Novel Approaches

Diffusion barriers are universal solutions for cells to achieve distinct organizations, compositions, and activities within a limited space. The influence of diffusion barriers on the spatiotemporal dynamics of signaling molecules often determines cellular physiology and functions. Over the years, the passive permeability barriers in various subcellular locales have been characterized using elaborate analytical techniques. In this review, we will summarize the current state of knowledge on the various passive permeability barriers present in mammalian cells. We will conclude with a description of several conventional techniques and one new approach based on chemically inducible diffusion trap (CIDT) for probing permeable barriers.