Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. In collaboration with the laboratory of Tamara Kinzer-Ursem, we have demonstrated that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of developing and juvenile mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles that enable newly synthesized proteins to be enriched for biochemical analyses using bead-based purification techniques or reacted with fluorophores to resolve the spatial distribution within tissues. This new tool will enable future studies that seek to quantify the composition and turnover of the murine proteome during growth, disease and repair.
Light scattering lipids and refractive index mismatches complicate imaging of cells and ECM within intact tissues. To overcome these challenges, my laboratory has been utilizing clearing methods that enhance visualization while maintaining specimen architecture. SeeDB is a simple water-based clearing agent that takes advantage of saturated fructose solutions having a refractive index close to that of fixed tissue (n~1.50) and permits imaging to depths greater than 2,000μm using conventional confocal microscopy. We have shown that SeeDB is useful for imaging tissue structure in dense musculoskeletal tissues.
While SeeDB greatly enhances the visualization of cells, the architecture of the ECM in developing limbs cannot be resolved in adequate detail. To address this limitation, my lab created a technique that greatly enhances visualization of the 3D architecture of developing tissues. An interpenetrating hydrogel network is formed throughout, which does not cross-link to proteins within the embryo. The cells are removed with detergent and the hydrogel provides a framework that maintains the 3D geometry of the ECM.
Quantification of the influence of ECM composition on tissue mechanics
Dr. Sarah Calve, Dr. Adrian Buganza Tepole, Dr. Corey Neu, Julian Jimenez, Kaitlin McCreery, Hannah Larson, Callan Luetkemeyer
The biomechanical properties of the ECM play an important role in cell migration, gene expression, and differentiation. The material properties of musculoskeletal tissues at the cellular level are usually measured on cryotomed (frozen) sections, which precludes the ability to study cell/matrix interplay due to disruptions in cell viability and tissue architecture from freeze-thaw cycling. To address this issue, we developed a technique to map the stiffness of living cells and surrounding matrix by atomic force microscopy in collaboration with the laboratory of Corey Neu. Our technique reveals significant differences between vibratomed (living) and cryotomed tissues and has the resolution to measure perturbations in cell and matrix stiffness due to biochemical and genetic disruptions in ECM organization.
Our previous work demonstrated that hyaluronic acid (HA) is upregulated during the early stages of muscle regeneration in newts and repair in the mouse. In vitro studies revealed HA significantly enhances migration and inhibits differentiation of skeletal muscle myoblasts, whereas laminin and Matrigel, which more closely mimic the ECM of undamaged skeletal muscle, promote differentiation. Furthermore, our results indicate that the way in which muscle cells respond to changes in stiffness is dependent on the extracellular matrix environment. Currently, we are investigating how HA regulates muscle cell migration and assembly during murine forelimb development. The data from these investigations are being used to design HA hydrogels of physiologically relevant stiffness to direct skeletal muscle behavior, in collaboration with the lab of Alyssa Panitch.