Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Wednesday Speaker Abstracts

Cutting the Tension with a Laser: Biophotonic Dissection of Stress Fibers and Contractile Signaling Sanjay Kumar . University of California, Berkeley, Berkeley, USA. The ability of the actomyosin cytoskeleton to distribute tensile forces at the cell-matrix interface at specific times and places is now understood to be critical to shape stabilization, motility, tissue assembly, and fate determination. I will discuss efforts my research group has been making to investigate the tensile properties of single stress fibers in living cells using single-cell biophotonic tools. Specifically, we have used femtosecond laser nanosurgery to spatially map the viscoelastic properties of actomyosin stress fibers and found that specific stress fiber compartments exhibit distinct viscoelastic properties that strongly depend on their subcellular location. In an effort to understand how these variations in viscoelastic properties translate into differences in tensile signaling and shape stability contributions, we have combined this method with fluorescence energy resonance transfer (FRET) probes of molecular tension within focal adhesions. These studies reveal that individual stress fibers can distribute tensile loads across a surprisingly large ensemble of adhesions, which is in turn related to the network connectivity of the individual fiber. Finally, we have begun to investigate the relative contributions of myosin activators (MLCK, ROCK) and isoforms (myosin IIA, IIB) to regulating tension within these pools of stress fibers, and we have coupled these measurements with single-cell micropatterning to investigate how viscoelastic properties are related to stress fiber geometry, inter-adhesion distances, and sarcomere/dense body architecture. Engineering Spatial Gradient of Signaling Proteins Using Magnetic Nanoparticles Zoher Gueroui . Ecole Normale Supérieure - CNRS, Paris, France. Intracellular biochemical reactions are often localized in space and time, inducing gradients of enzymatic activity that may play decisive roles in determining cell’ s fate and functions. However, the techniques available to examine such enzymatic gradients of activity remain limited. Here, we propose a new method to engineer a spatial gradient of signaling protein concentration within Xenopus egg extracts using superparamagnetic nanoparticles. We show that, upon the application of a magnetic field, a concentration gradient of nanoparticles with a tunable length extension is established within confined egg extracts. We then conjugate the nanoparticles to RanGTP, a small G-protein controlling microtubule assembly. We found that the generation of an artificial gradient of Ran-nanoparticles modifies the spatial positioning of microtubule assemblies. Furthermore, the spatial control of the level of Ran concentration allows us to correlate the local fold increase in Ran nanoparticle concentration with the spatial positioning of the microtubule-asters. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. More generally, these results show how magnetic nanoparticles and magnetogenetic tools can be used to control the spatiotemporal dynamics of signaling pathways.

32