Shaohua Hu

Filamin A Is Essential for Active Cell Stiffening but not Passive Stiffening under External Force

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.

Cell spreading controls balance of prestress by microtubules and extracellular matrix.

The controversy surrounds the cellular tensegrity model. Some suggest that microtubules (MTs) must bear a significant portion of cell contractile stress (prestress) if tensegrity is a useful model. Previously we have shown that for highly spread airway smooth muscle cells (areas>2500 microm2) MTs balance a significant but small potion (average 14%) of the prestress. To further explore if controlling the degree of cell spreading could modulate the portion of the prestress balanced by MTs, we utilized a recent method by which tractions are quantified in cells that are constrained within micropatterned adhesive islands of defined sizes on the surface of flexible polyacrylamide gels containing fluorescent microbeads. The prediction is that if MTs balance a portion of the contractile stress, then, upon their disruption, the portion of the stress balanced by MTs would shift to the substrate, causing an increase in traction and strain energy. We first activated the cells maximally with histamine and then disrupted the MTs with colchicine. Histamine resulted in an increase in intracellular calcium whereas ensuing colchicine addition in the presence of histamine did not change intracellular calcium concentration, suggesting there was no additional net increase in contractile stress inside the cell. We found that following disruption of MTs the increase in traction and strain energy varied with the degree of cell spreading: as the cell projected areas increased from 500 micrometer 2 to about 1800 micrometer 2, the percent increase in tractions decreased from 80% to about a few percent and the percent increase in strain energy decreased from 200% to almost zero percent, indicating the portion of the prestress balanced by MTs decreased as the cells increased spreading. These findings demonstrate that complementary role of the extracellular matrix and the MTs in balancing the prestress is controlled by the degree of cell spreading.

Imaging Stress Propagation in the Cytoplasm of a Living Cell

A fundamental issue in mechanotransduction is to determine pathways of stress propagation in the cytoplasm. We describe a recently developed synchronous detection approach that can be used to map nanoscale distortions of cytoskeletal elements and nuclear structures in living individual cells using green fluorescent protein technology and 3D magnetic twisting cytometry. This approach could be combined with single-cell biochemical and biological assays to help elucidate mechanisms of mechanotransduction.

Displacement field of the cytoskeleton in response to a local load

Mechanical stresses acting on the apical cell surface are transmitted to the anchoring sites of the cell via cytoskeletal polymers, but details of the stress-, strain- or deformation field within the cell are largely unknown. Here we have measured the deformation field within cultured smooth muscle cells in response to small stresses. Stresses were applied to integrin receptors on the cell surface via magnetic microbeads (4.5 /spl mu/m diameter). The beads were torqued in a sinusoidally varying magnetic twisting field (specific torque amplitude of 90 Pa, frequency 0.3 Hz). Cells were transfected to express either fluorescent mitochondria, microfilaments, or microtubules. Ten images were taken during each of ten or more twisting cycles, from which we computed the deformation field within the cell. Our results confirm that mechanical stresses in cells can be transmitted via focal adhesions on the apical cell surface to the internal cytoskeleton. Importantly, cytoskeletal deformations in most cells decayed to below the resolution limit within a short distance (/spl sim/5/spl mu/m) from the locus of stress application.