36 / 88
32
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.