What holds us together? Examining the protein building blocks of our tissues, one at a time
Collagen is the fundamental structural protein in vertebrates and is widely used as biomaterial, for example as a substrate for tissue engineering. Assembled from individual triple-helical proteins to make strong fibres, collagen is a beautiful example of a hierarchical self-assembling system. In spite of its prevalence and mechanical importance in biology, surprisingly little is known about how or even whether the mechanics of the triple helix vary within its sequence. The flexibility of the entire triple helix is also unresolved, as is its response to stress. In this presentation, I will describe the tale of collagen’s mechanics that my research group is unravelling using a variety of single-molecule analysis techniques (optical tweezers, atomic force microscopy and centrifuge force microscopy). These complementary single-molecule studies are providing new insight into the molecular basis for mechanical response.
Raman spectroscopy for use in detection of ionizing radiation damage in bimolecular systems
Radiation therapy is currently used to treat up to 50% of new cancer cases. Despite the widespread use of radiation to treat cancer, the ability to monitor patient response during treatment remains limited. Hence, even though radiation therapy is typically delivered in daily fractions, currently, fractions are not optimized to individual patient response to treatment. Raman spectroscopy is an optical spectroscopic technique that provides a wealth of molecular-level information on an interrogated system. We here employ Raman spectroscopy to monitor cellular and tissue response to ionizing radiation used in cancer therapy. We show that the technique has the ability to monitor, for example, radiation-induced glycogen production both at the cellular and full tissue level. The results indicate that Raman spectroscopy may be a valuable tool in the current drive for personalized radiation therapy.
Development and characterization of sensitive/selective sensors on integrated lab-on-chip applications
Portable sensors and biomedical devices are influenced by high precision control of microfluidic systems, low-cost fabrication techniques, detection and analysis capabilities. The integration of sensing devices into the chip is still a major problem in microfluidic devices. This presentation focuses on design and fabrication of a precise liquid handling system for flow-thru biosensors using open and closed digital microfluidic (DMF) systems. Real-time on-chip detection of biological species in the biosensor is demonstrated using both optical detection of individual stained cells as well as measuring capacitance variation of a cluster of biological cells passing through the readout site. Adding sample preparation, filtering and purification sites, the proposed biosensor can be used for total analysis or single cell analysis assays. Featuring low-cost hardware with high capacitance measurement resolution and rapid chip fabrication techniques, the proposed biosensor design has the potential to be commercialized as viable solution for life-science research and clinical diagnostics.
Mapping cell surface adhesion by single-cell rotation tracking and adhesion footprinting
Rolling adhesion is the behaviour that leukocytes and circulating tumour cells exhibit as they passively roll along blood vessel walls under flow. It plays a critical role in capturing cells in the blood, guiding them toward inflammation sites, and activating cell signalling pathways to enable their subsequent transmigration. Rolling adhesion is mediated by catch-bond-like interactions between selectins expressed on endothelial cells lining blood vessels and P-selectin glycoprotein ligand-1 (PSGL-1) found at microvilli tips of leukocytes. Despite our understanding of individual components of this process, how the molecular details of adhesion bonds scale to cell-surface adhesion and rolling behaviour remains poorly understood. Here, we developed 2 methods that map the functional adhesion sites and their strength on a leukocyte surface. The first method relies on tracking the rotational angle of a single rolling cell, which confers advantages over standard methods that track the centre-of-mass alone. Constructing the adhesion map from the instantaneous angular velocity reveals that the adhesion profile along the rolling circumference is inhomogeneous. We corroborated these findings with a second method that allowed us to obtain a footprint of molecular adhesion events using DNA-based molecular force probes. Our results reveal that adhesion at the functional level is not uniformly distributed over the leukocyte surface as previously assumed, but is instead patchy.
Molecular Probes to Study the Ca2+ Signaling Cascade in Neurons and Astrocytes
In the brain, a calcium signaling cascade in astrocytes correlates with the firing of neurons at synapses. The exact function of this Ca2+ cascade remains elusive—mostly due to the lack of tools to study it in live cells. We report the design and use of a set of molecular probes to visualize, label, and influence Ca2+ proteins involved in the signaling cascade in their native environment.
Using organic synthesis, we have modified ligands displaying exquisite selectivity for specific Ca2+ ion channels. The ligands include: domoic acid, thapsigargin and ryanodine. They were initally conjugated with fluorescent tags to enable their study in live cells. Using inhibitors and agonists allows us to both monitor and systematically control Ca2+ ions’ entry into cells, signal amplification, and intracellular depletion events. We then characterize the dynamics of Ca2+ signaling events in cultured astrocytes using combined imaging and electrophysiology techniques. Thus, we apply the molecules to deconvolute the microscopic events underpinning the communication between neurons and glia.
Advantages of our approach over existing genetic methods include the ability to monitor and perturb gating events simultaneously. Though we chose to study astrocytes for the exciting questions they pose, the ubiquity of Ca2+ ion channels makes these probes broadly applicable to the study of Ca2+ signaling in other tissues.