We aim to understand the physical characteristics of actin network growth through direct measurements of both force generation and network growth velocity. Measurements of force and velocity are important biophysical parameters in understanding the fundamental physics of cellular motility. We measure network growth velocity and force production of reconstituted actin networks using a custom-built AFM equipped with epi-fluorescence. In addition to measurements of force and velocity, we are interested in how the mechanical properties of a growing actin network may play a role in how cells respond to their physical environment.

Optical trapping experiments on actin

Growing, cross-linked networks of polymerizing actin filaments drive cell protrusions in many eukaryotic cells and propel intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii. While chemical signals are known to guide network growth in cellular processes such as chemotaxis, the extent to which external forces can also influence the direction of motion has not been studied. We directly quantify and manipulate the curvature of paths taken by actin-propelled microspheres in three dimensions using an optical trap. The magnitude of path curvature yields information about the number of actin filaments that interact with a microsphere at any one time. In addition, the variation in vector curvature indicates that actin filament dynamics play a role in force generation with a characteristic time scale. Finally, we can use tiny optical forces to bias the direction of movement and the network curvature. These forces are considerably smaller than current estimates of the total pushing forces of growing actin networks, implying a large mechanical advantage that may be used by cells to guide network growth.

Phase separation of membrane with membrane associated actin network

Spatial organization of cell membrane underlies many important cellular functions, in particular signal transduction. A popular theory is the raft hypothesis that suggests formation of cholesterol rich lipid domains can be driven solely by characteristic lipid-lipid interactions. More recently, single molecular tracking on T-cells showed that membrane micro-domains can be created by protein-protein interactions. While the origin of spatial organization remains to be debated, both sides claim that actin cytoskeleton is involved. Synthetic lipid vesicles containing a saturated, an unsaturated lipids, and cholesteral have been shown to undergo phase separation, thus making it a useful model system to study raft-like phenomenon.

Cytoskeletal association to membrane can occur through phosphatidylinoside 4,5-bisphosphate activation of nucleation promoting factor, which activates assembly of a dendritic actin network. We are studying the effect of membrane associated actin on phase separation behavior of the lipid membrane. One common observable of a system capable of phase separation is to measure the temperature (miscibility temperature) at which phase separation occurs. By measuring the miscibility temperature in the absence/presence of membrane associated actin, we hope to define a better role of actin cytoskeleton on membrane organization.

A giant unilamellar vesicle made of DOPC, DPPC, and cholesterol with trace amount of fluorescent dye is shown here to undergo phase separation at the miscibility transition temperature of 31° C.