The BEAMS Lab focuses on the relationship between mechanical stresses on cells and their microenvironment. The use of laser tweezers is integral in evaluating and influencing the microenvironment of a cell.
As such, many projects associated with the BEAMS Lab involve the use of laser tweezers in capturing microparticles and using them as a means to assess local microenvironment mechanics and to induce forces onto a cell and its environment.
Our laboratory is studying the role of physical forces for activation of the Notch receptor. The Notch receptor is curious in its general requirement for ligand endocytosis for generating Notch signals. That is to say, ligand presented on the signal-sending neighboring cell, must be endocytosed by that cell to activate Notch. This so called pulling force model is a special case of mechanotransduction for which mechanical forces appear required for signaling, but the cells are not probing their mechanical microenvironment. The figure below illustrates this pulling force model as put forth by our collaborator Professor Gerry Weinmaster (retired) formally of UCLA.
Together we demonstrated that Notch signaling was dependent up Mib-mediated ligand ubiquitylation, the endocytic machinery components clatherin, dynamic, and epsins, as well as actin polymerization inside of the ligand cell. This finding suggests that the endocytic machinery in a ligand cell applies physical force through a Notch-ligand interaction to unfold notch for signaling. Curiously, we found that while the rupture force of the Notch-ligand interaction was independent on these factors (Shergill et al.), the ability of the ligand to generate force was dependent on these factors (Meloty-Kapella et al.).
We are currently studying the mechanical work required for Notch activation
We use microrheology to map the local stiffness of the ECM in 3D models of capillary morphogenesis. This work is in collaboration with the laboratory of Andrew Putnam at the University of Michigan.
E. Kniazeva; J.W. Weidling; R. Singh; E.L. Botvinick; M.A. Digman; E. Gratton; A.J. Putnam
Quantification of local matrix deformations and mechanical properties during capillary morphogenesis in 3D
Integrative Biology. 4 (4), 431-439.
M.A. Kotlarchyk; S.G. Shreim; M.B. Alvarez-Elizondo; L.C. Estrada; R. Singh; L. Valdevit; E. Kniazeva; E. Gratton; A.J. Putnam; E.L. Botvinick
Concentration independent modulation of local micromechanics in a fibrin gel.
PLoS One. 6 (5), e20201.
The following reflection confocal microscopy video shows a HUVEC-derived capillary invading a 3-D fibrin hydrogel. Notice the local deformations to the matrix as the tip cell contracts. We aim to determine the local resistance to such deformations, and the work generated by the cell:
The video below is for the same model, but here you can see a microbead embedded within the hydrogel. We can physically oscillate that bead with light to measure the surrounding stiffness. In our experiments, we typically have a microbead every 50 microns or less away from its nearest neighbor so we can reconstruct the mechanical microenvironment.
Our lab is working with renowned breast cancer scientist Thea Tlsty and theoretical physicist Clare Yu to investigate the roles of stromal stiffness in the progression of malignant breast cancer. We are currently investigating in vitro models of breast cancer combining naturally derived matrices composed of reduced basal membrane proteins and collagen I, with our stiffening device and microrheology measurements of local stiffness. Using our stiffening device we can tune the stiffness of Matrigel from 50Pa to well over 1 kPa in the same dish. We are combining this tuning of stiffness with nonlinear imaging techniques such as CARS and multiphoton fluorescence microscopy to correlate cell morphology and molecular dynamics in response to local changes in stiffness – in real time.
Currently, no methods exist for the screening of cellular mechanotransduction at high-throughput. While sophisticated imaging technologies are readily available for the rapid assessment of cellular activity, they cannot be coupled easily coupled with existing technologies to apply precise mechanical perturbation. Thus, we aim to provide a novel method for high-throughput assessment of cellular mechanotransduction using laser generated cavitation bubbles.
Compton, Jonathan L., Justin C. Luo, Huan Ma, Elliot Botvinick, and Vasan Venugopalan. “High-Throughput Optical Screening of Cellular Mechanotransduction.” Nature Photonics 8, no. 9 (August 3, 2014): 710–15. doi:10.1038/nphoton.2014.165.
Our The Shear Gradient Device (SGD) increases the stiffness of naturally derived extracellular matrices (ECMs). All ECM hydrogels exhibit strain hardening (panel A). For example, fibrin stiffness increases linearly but weakly with low values of strain, while at higher strains it nonlinearly stiffens . This observation inspired our innovative SGD, in which an ECM gel is non-uniformly stretched from 0% to over 100%. The device, shown in panel B and described in , comprises a microscope stage plate machined to hold a 35 mm glass-bottom Petri dish (red) containing a cylindrical post (teal) held in place by a cantilevered arm housing a leadscrew (silver), spring plunger assembly (black) and lever arm (concealed by cantilever) all designed to delicately and precisely rotate the post. An ECM hydrogel is first polymerized within the dish where it binds to the dish and the post. The post is then rotated and held in position thus shearing the gel. Regions nearest to the post experience the most shear strain while regions nearest to the wall of the dish experience the least. The post can be offset to increase the range of strain within the gel, as shown in panel B. Finite element analysis (FEA) of the predicted shear strain is shown in panel C. The pseudocolor representation indicates near-zero strain at the bottom of the dish (blue) and strains in excess of 13% near the post. To directly measure the heterogeneity in stiffness imposed by our shear gradient, we performed active microrheology (AMR) on a fibrin gel (2.5 mg/ml) in regions near to the post as indicated in panel D. We found steep radial and circumferential gradients in stiffness near to and out to approximately 1mm distance from the post. Measurement of stiffness before and after rotation of the post shows as much as a 10-fold stiffening in regions near the post and no stiffening in regions away from the post  (data not shown). Our group has recently completed a study of Matrigel stiffening (paper in preparation). Even at a very low concentration of Matrigel (3.3 mg/ml), we could tune stiffness from 10 Pa to over 500 Pa within a single petri dish. To put our finding in context, Matrigel stiffness spanned the range between normal and malignant breast tissue, as reported .
1. Brown, A.E., et al., Multiscale mechanics of fibrin polymer: gel stretching with protein unfolding and loss of water. Science, 2009. 325(5941): p. 741-4.
2. Kotlarchyk, M.A., et al., Concentration Independent Modulation of Local Micromechanics in a Fibrin Gel. PLoS One, 2011. 6(5): p. e20201.
3. Levental, K.R., et al., Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling. Cell, 2009. 139(5): p. 891-906.