Renner_D_Lrg_cellstretch_device
- 1. Design and Fabrication of Large-Scale Cell Stretch Device for Mass Spectrometry Derrick Renner and Soichiro Yamada Department of Biomedical Engineering, University of California, Davis, CA 1. Design and fabrication of large-scale cell-stretch device Ueda S, Blee AM, Macway KG, Renner DJ, Yamada S (2015) Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase. PLoS ONE 10(3): e0122886. doi: 10.1371/journal.pone.0122886 Figure 1: Large-scale cell-stretching device. (Top) Design in SolidWorks, (Bottom) Cell-stretching device with media and cells • Sufficient capacity for mass spectrometry protein analysis • Reusable and sterilizable • User friendly, minimal amount of tools required for use • Precise, electronically-controlled stretching 2. Cellular responses to mechanical strain 4. Biochemical analysis of force-sensitive protein complexes 3. Principle of proximal biotinylation to detect force- sensitive protein complexes Small-scale Large-scale Figure 2: Cellular morphology. (Left): Unstretched cells show typical cellular morphology, (Right): Stretched cells (25% stretch in direction of arrow) have an elongated morphology. Figure 4: Biochemical analysis of force- sensitive protein complexes using Western Blot. (A) Western Blot of small (4cm2 plating area) stretch chamber. (B) Relative band intensities of unstretched (control) and stretched (stretch) samples. βcat (n = 4), αcat (n = 4) and MyoIIA (n = 7). Results were analyzed using a one-way ANOVA; significance was determined using Dunnett’s post hoc test. Results were considered significant with P<0.05. (C) Western Blot of large-scale cell-stretching device. Stretching and Cellular Morphology: • Two cell-plating areas of 84.5 cm2 each (max, unstretched). • More than 20 times the area of original stretch chambers • Acrylic and stainless steel components can be sterilized • Full assembly requires one Phillips screwdriver and one hex wrench • Servo motor provides adjustable stretch lengths and rates. In our previous study (Ueda et. al 2015), we have shown that stretching of the cell monolayer induces the biotinylation of myosin IIA by BirA* tagged α-catenin. However, the previous study was hampered by the use of small cell stretch chambers that limited the quantity of isolated proteins sufficient for a large scale proteomic analysis. Therefore, our goal is to scale-up the cell stretch chambers and validate our new design with our previous results. CA Figure 3: Characterization of promiscuous BirA (BirA*)-tagged α-catenin expressing cells. (A) The illustration shows in situ proximal biotinylation and subsequent purification of biotinylated proteins along with a schematic of a BirA*-α-catenin construct. (B) Localization of BirA*-α-catenin and streptavidin-specific labeling to cell-cell contacts; the latter demonstrates the proximal biotinylation effect of BirA*-α-catenin (with biotin present). Scale bar in bottom right window is 20 μm. • BirA* exhibits promiscuous biotinylation activity through its rapid release of biotin donor, bioAMP, which non-specifically reacts with nearby primary amines • BirA* sequence was inserted between GFP and α-catenin (Fig 3A) and stably expressed in MDCK cells • The BirA*-α-catenin proteins localized to cell-cell contacts in MDCK cells (Fig 3B) • In biotin-containing media, streptavidin specific labeling localized to cell-cell contacts, demonstrating the proximal biotinylation by BirA*-α-catenin A B • Unstretched cells (control) have a cobblestone geometry • Cells were subjected to an oscillating stretch, at a rate of approximately 2.5 cycles/minute and a magnitude of 25% of the original PDMS substrate length. • Stretched cells formed an elongated morphology perpendicular to the direction of stretch (Fig 2). Cadherin-mediated cell-cell interaction is a critical requirement for the development of multicellular organisms. The cadherins, a family of cell-cell adhesion proteins, play fundamental roles in cell organization during physiological processes such as tissue growth and skin regeneration, and the abnormal regulation of cadherins often leads to pathological processes such as cancer metastasis and formation of leaky blood vessels. A large-scale cell stretching device was designed in SolidWorks and fabricated in a machining laboratory. This device consists of two lanes: one fixed lane (control) and one servomotor- controlled stretching lane. The large surface provides the substrate required for a large number of cells to adhere. This, in turn, enables us to isolate a large quantity of cells, thus proteins, sufficient for mass spectrometry. Abstract Publications Device fabrication: UC Davis Biomedical Engineering TEAM Lab – Steven Lucero Yamada Lab – Mary Sedarous, Katherine Macway, Brent Weyers, Makena Ewald, Shuji Ueda, and Alexandra Blee Funding: NIH Eureka GM094798 (SY) and Kobe University Institutional Program for Young Researcher Overseas Visits (SU) Introduction Acknowledgements Results • While the biotinylation of α-catenin and β-catenin remained similar regardless of stretch, the biotinylation of Myosin IIA increased in mechanically stretched samples. • Myosin IIB does not appear to be biotinylated in any of the assays. • Small-scale and large-scale biochemical analysis of biotinylation yielded similar results B During embryogenesis or wound healing, neighboring cells maintain contact and migrate collectively. Due to constant pulling and pushing between migrating neighboring cells, we hypothesize that mechanical forces regulate the interaction between the cell-cell adhesion complex and the actin cytoskeleton, therefore, regulating the adhesive strength. To identify force-sensitive protein complexes at cell- cell junctions, our innovative biochemical analysis combines in situ proximal biotin labeling with a cell stretch device that promotes the formation of force-sensitive complexes. By fusing α-catenin with a promiscuous biotin ligase, any proximal proteins of α-catenin will be biotinylated. Our preliminary study demonstrates that α- catenin and myosin IIA are likely interacting in a force-dependent manner. While the current approach is suited for the candidate screening of force-sensitive proteins, the application of proteomic screening to this approach will be transformative, because the proteomic screening will reveal the total composition of α-catenin associated proteins in the presence or absence of external forces, a critical first step in deciphering the molecular basis of mechano-transduction. However, the key limitation of the current protocol is the small size of the cell stretch chambers, which limits the quantity of protein samples. Our goal is to re-design and scale-up the current protocol to isolate the quantity of purified proteins sufficient for mass spectrometry and identify the force-sensitive complex surrounding α-catenin. Conclusion and Future Work When mechanical stress is applied to a cell monolayer, cell-cell junctions stiffen and recruit proteins such as actin and vinculin, suggesting the formation of a force-sensitive complex at these force-bearing cell junctions. Since these interactions are weak and transient, these type of force-induced protein-protein interactions are impossible to identify using traditional biochemical analysis, which requires stable protein interactions for isolation. To uncover the types of proteins involved in force- sensitive protein complexes, we designed an assay based on in situ proximal biotinylation. Our approach will identify a set of potential proteins involved in these transient, force-sensitive complexes. Figure 3. At the sites of cell-cell contact, cadherin junctions recruit and alter the actin network. This cell-cell adhesion induced actin network is thought to provide the forces and strength that are necessary for collective cell migration. A mutant biotin ligase, BirA* is linked to α-catenin, a well-known binding partner in the cadherin complex. BirA* biotinylates the primary amines of proximal proteins; this labels the proteins nearby the cadherin complex with biotin. Subsequently, the biotinylated proteins are purified using streptavidin beads and identified through a western blot. Materials & Methods The results of this trial are consistent with our previous study, demonstrating an increase in biotinylation of myosin IIA upon cell stretching. This validates the use of the large scale proximal biotinylation as a methodology to identify force-sensitive protein interactions. In order to obtain a comprehensive list of the force-sensitive proteins involved in cadherin-mediated cell-cell adhesion, we plan on using mass spectrometry in conjunction with the large-scale cell-stretching device. Design Specifications: Prototype Specifications: Comparison of small and large cell-stretching devices