Vimentin supports cell polarization by enhancing centrosome function and microtubule acetylation.

Cell polarity is important for controlling cell shape, motility and cell division processes. Vimentin intermediate filaments are important for cell migration and cell polarization in mesenchymal cells and assembly of vimentin and microtubule networks is dynamically coordinated, but the precise details of how vimentin mediates cell polarity remain unclear. Here, we characterize the effects of vimentin on the structure and function of the centrosome and the stability of microtubule filaments in wild-type and vimentin-null mouse embryonic fibroblasts. We find that vimentin mediates the structure of the pericentriolar material, promotes centrosome-mediated microtubule regrowth and increases the level of stable acetylated microtubules in the cell. Loss of vimentin also impairs centrosome repositioning during cell polarization and migration processes that occur during wound closure. Our results suggest that vimentin modulates centrosome structure and function as well as microtubule network stability, which has important implications for how cells establish proper cell polarization and persistent migration.

Spreading rates of bacterial colonies depend on substrate stiffness and permeability.

The ability of bacteria to colonize and grow on different surfaces is an essential process for biofilm development. Here, we report the use of synthetic hydrogels with tunable stiffness and porosity to assess physical effects of the substrate on biofilm development. Using time-lapse microscopy to track the growth of expanding Serratia marcescens colonies, we find that biofilm colony growth can increase with increasing substrate stiffness, unlike what is found on traditional agar substrates. Using traction force microscopy-based techniques, we find that biofilms exert transient stresses correlated over length scales much larger than a single bacterium, and that the magnitude of these forces also increases with increasing substrate stiffness. Our results are consistent with a model of biofilm development in which the interplay between osmotic pressure arising from the biofilm and the poroelastic response of the underlying substrate controls biofilm growth and morphology.

Vimentin intermediate filaments modulate cell traction force but not cell sensitivity to substrate stiffness.

The ability of cells to sense and respond to the mechanical stiffness of the surrounding matrix is important to support normal cell function, wound healing, and development. Central to the process of durosensing is the cytoskeleton composed of three classes of filaments: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that contributes significantly to cell mechanics and cell traction force, which is required to probe extracellular matrix. The role of vimentin in how cells sense and respond to the mechanical rigidity of extracellular matrix is largely unclear. To investigate the role of vimentin in durosensing, we knocked down the vimentin expression level in 3T3 fibroblasts using shRNA transfection and measured cellular responses as functions of substrate stiffness. We quantified durosensitivity by the rates at which cell area and traction force change with substrate stiffness. Our results show that that vimentin plays a role in durosensing by modulating traction force and knocking out vimentin did not significantly affect durosensitivity. These results indicate that vimentin may be a redundant component of the machinery that cells use to sense substrate stiffness.

Reliable and mobile all-fiber modular optical tweezers.

Miniaturization and integration of optical tweezers are attractive. Optical fiber-based trapping systems allow optical traps to be realized in miniature systems, but the optical traps in these systems lack reliability or mobility. Here, we present the all-fiber modular optical tweezers (AFMOTs), in which an optical trap can be reliably created and freely moved on a sample substrate. Two inclined optical fibers are permanently fixed to a common board, rendering a modular system where fiber alignments are maintained over months. The freely movable optical trap allows particles to be trapped in their native locations. As a demonstration, we applied AFMOTs to trap and deform freely floating individual cells. By the cell mechanical responses, we differentiated the nontumorigenic breast epithelial cell line (MCF10A) from its cancerous PTEN mutants (MCF10 PTEN-/-). To further expand the functionalities, three modalities of AFMOTs are demonstrated by changing the types of fibers for both the optical trap creation and particle position detection. As a miniature and modular system that creates a reliable and mobile optical trap, AFMOTs can find potential applications ranging from point-of-care diagnostics to education, as well as helping transition the optical trapping technology from the research lab to the field.

Objective-lens-free Fiber-based Position Detection with Nanometer Resolution in a Fiber Optical Trapping System.

Position detection with high accuracy is crucial for force calibration of optical trapping systems. Most existing position detection methods require high-numerical-aperture objective lenses, which are bulky, expensive, and difficult to miniaturize. Here, we report an affordable objective-lens-free, fiber-based position detection scheme with 2 nm spatial resolution and 150 MHz bandwidth. This fiber based detection mechanism enables simultaneous trapping and force measurements in a compact fiber optical tweezers system. In addition, we achieved more reliable signal acquisition with less distortion compared with objective based position detection methods, thanks to the light guiding in optical fibers and small distance between the fiber tips and trapped particle. As a demonstration of the fiber based detection, we used the fiber optical tweezers to apply a force on a cell membrane and simultaneously measure the cellular response.