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In JoVE (1)
Other Publications (8)
- Developmental Neurobiology
- Nano Letters
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Structural Biology
- Journal of the American Chemical Society
- Materials Research Society Symposia Proceedings. Materials Research Society
- Lab on a Chip
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
Articles by Lance C. Kam in JoVE
JoVE General
Microcontact Printing of Proteins for Cell Biology
Department of Biomedical Engineering, Columbia University
Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.
Other articles by Lance C. Kam on PubMed
Local Presentation of L1 and N-cadherin in Multicomponent, Microscale Patterns Differentially Direct Neuron Function in Vitro
The ability to pattern multiple bioactive cues on a surface is valuable for understanding how neurons interact with their complex extracellular environment. In this report, we introduce a set of methods for creating such surfaces, with the goals of understanding how developing neurons integrate multiple biologically relevant signals and as a tool for studying interactions between multiple neurons. Multiple microcontact printing steps are combined on a single surface to produce an array of polylysine nodes, interconnected by lines of proteins based on the extracellular domains of L1 or N-cadherin. Surprisingly, the N-cadherin protein could also be directly printed onto surfaces while retaining its biological activity. Rat hippocampal neurons selectively attached to the polylysine nodes, differentially extending axonal and dendritic processes along the patterns of L1 and N-cadherin, thus demonstrating control over neuron attachment and outgrowth. Combining these three biomolecules on a single surface revealed a highly complex pattern of protein recognition. Dendrites extended exclusively on N-cadherin patterns, while axons exhibited a very high degree of selectivity on L1 patterns, preferentially at distances greater than 55 mum from the cell body. At shorter distances, axonal processes recognized both L1 and N-cadherin, revealing a new aspect of neuron polarity and axon specification. This onset of L1 selectivity correlated with the establishment of intracellular L1 polarity, suggesting a functional outcome of the process of neuron polarization that has implications in development of neural tissues and creation of in vitro neuron networks.
Non-Brownian Diffusion of Membrane Molecules in Nanopatterned Supported Lipid Bilayers
Molecules associated with the outer surface of living cells exhibit complex, non-Brownian patterns of diffusion. In this report, supported lipid bilayers were patterned with nanoscale barriers to capture key aspects of this anomalous diffusion in a controllable format. First, long-range diffusion coefficients of membrane-associated molecules were significantly reduced by the presence of the barriers, while short-range diffusion was unaffected. Second, this modulation was more pronounced for large molecular complexes than for individual lipids. Surprisingly, the quantitative effect of these barriers on long-range lipid diffusion could be accurately simulated using a simple, continuum-based model of diffusion on a nanostructured surface; we thus describe a metamaterial that captures the properties of the outer membrane of living cells.
Micropatterning of Costimulatory Ligands Enhances CD4+ T Cell Function
Spatial organization of signaling complexes is a defining characteristic of the immunological synapse (IS), but its impact on cell communication is unclear. In T cell-APC pairs, more IL-2 is produced when CD28 clusters are segregated from central supramolecular activation cluster (cSMAC)-localized CD3 and into the IS periphery. However, it is not clear in these cellular experiments whether the increased IL-2 is driven by the pattern itself or by upstream events that precipitate the patterns. In this article, we recapitulate key features of physiological synapses using planar costimulation arrays containing antibodies against CD3 and CD28, surrounded by ICAM-1, created by combining multiple rounds of microcontact printing on a single surface. Naïve T cells traverse these arrays, stopping at features of anti-CD3 antibodies and forming a stable synapse. We directly demonstrate that presenting anti-CD28 in the cell periphery, surrounding an anti-CD3 feature, enhances IL-2 secretion by naïve CD4(+) T cells compared with having these signals combined in the center of the IS. This increased cytokine production correlates with NF-kappaB translocation and requires PKB/Akt signaling. The ability to arbitrarily and independently control the locations of anti-CD3 and anti-CD28 offered the opportunity to examine patterns not precisely attainable in cell-cell interfaces. With these patterns, we show that the peripheral presentation of CD28 has a larger impact on IL-2 secretion than CD3 colocalization/segregation.
Capturing the Nanoscale Complexity of Cellular Membranes in Supported Lipid Bilayers
The lateral mobility of cell membranes plays an important role in cell signaling, governing the rate at which embedded proteins can interact with other biomolecules. The past two decades have seen a dramatic transformation in understanding of this environment, as the mechanisms and potential implications of nanoscale structure of these systems has become accessible to theoretical and experimental investigation. In particular, emerging micro- and nano-scale fabrication techniques have made possible the direct manipulation of model membranes at the scales relevant to these biological processes. This review focuses on recent advances in nanopatterning of supported lipid bilayers, capturing the impact of membrane nanostructure on molecular diffusion and providing a powerful platform for further investigation of the role of this spatial complexity on cell signaling.
Self-aligned Supported Lipid Bilayers for Patterning the Cell-substrate Interface
Supported lipid bilayers capture the fluidity and chemical properties of cellular membranes. In this report, we introduce a method for creating surfaces that contain multiple, aligned regions of supported membranes of different compositions at scales of micrometers and smaller. This method uses the design of a diffusional barrier to increase the resolution that can be achieved directly using traditional bilayer patterning techniques, such as laminar flow. We demonstrate the use of this platform for presenting ligands to the T Cell Receptor and LFA-1 that are tethered to separate, closely juxtaposed regions of bilayer, capturing an important aspect of the natural organization observed between T cells and Antigen Presenting Cells. Our results present a novel platform for the study of spatial separation of extracellular ligands and its impact on cell signals.
Nanoengineering of Immune Cell Function
T lymphocytes are a key regulatory component of the adaptive immune system. Understanding how the micro- and nano-scale details of the extracellular environment influence T cell activation may have wide impact on the use of T cells for therapeutic purposes. In this article, we examine how the micro- and nano-scale presentation of ligands to cell surface receptors, including microscale organization and nanoscale mobility, influences the activation of T cells. We extend these studies to include the role of cell-generated forces, and the rigidity of the microenvironment, on T cell activation. These approaches enable delivery of defined signals to T cells, a step toward understanding the cell-cell communication in the immune system, and developing micro/nano- and material- engineered systems for tailoring immune responses for adoptive T cell therapies.
Combined Microfluidics/protein Patterning Platform for Pharmacological Interrogation of Axon Pathfinding
Assembly of functional neural circuits relies on the ability of axons to navigate a complex landscape of guidance cues in the extracellular environment. In this report, we investigate localized cell signaling in response to these cues by combining a microfabricated compartmentalization chamber with multicomponent, protein-micropatterned surfaces; this system offers improved spatial resolution and new capabilities for targeted manipulation of neuronal axons. We illustrate the potential of this system by addressing the role of fibroblast growth factor receptor (FGFR) signaling in modulating axon guidance by N-cadherin. Motor neurons that were derived from embryonic stem cells extend axons from one compartment through a microchannel barrier and into a second compartment containing patterns of N-cadherin, against a background of laminin. N-cadherin was effective in both guiding and accelerating motor axon outgrowth. Using the chamber system to target the application of pharmacological agents to specific parts of the neuron, we demonstrate that FGFR signaling in the axon but not the cell body increases the rate of axon outgrowth while not affecting guidance along N-cadherin. These results demonstrate that cell signaling must take into account the spatial layout of the cell. This new platform provides a powerful tool for understanding such effects over a wide range of signaling systems.
Concentration-dependent Requirement for Local Protein Synthesis in Motor Neuron Subtype-specific Response to Axon Guidance Cues
Formation of functional motor circuits relies on the ability of distinct spinal motor neuron subtypes to project their axons with high precision to appropriate muscle targets. While guidance cues contributing to motor axon pathfinding have been identified, the intracellular pathways underlying subtype-specific responses to these cues remain poorly understood. In particular, it remains controversial whether responses to axon guidance cues depend on axonal protein synthesis. Using a growth cone collapse assay, we demonstrate that mouse embryonic stem cell-derived spinal motor neurons (ES-MNs) respond to ephrin-A5, Sema3f, and Sema3a in a concentration-dependent manner. At low doses, ES-MNs exhibit segmental or subtype-specific responses, while this selectivity is lost at higher concentrations. Response to high doses of semaphorins and to all doses of ephrin-A5 is protein synthesis independent. In contrast, using microfluidic devices and stripe assays, we show that growth cone collapse and guidance at low concentrations of semaphorins rely on local protein synthesis in the axonal compartment. Similar bimodal response to low and high concentrations of guidance cues is observed in human ES-MNs, pointing to a general mechanism by which neurons increase their repertoire of responses to the limited set of guidance cues involved in neural circuit formation.
