Shunichi Usami

Visualizing the mechanical activation of Src

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin–cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean ± s.e.m.) of 18.1 ± 1.7 nm s-1. This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

The interaction of leukocytes and erythrocytes in capillary and postcapillary vessels

Abstract In capillaries white blood cells tend to flow with a lower velocity than red blood cells. This is due to the larger volume of the white cells and their spherical shape as compared to that of the red cells with their biconcave disk shape, as well as to the smaller deformation of the white cell during flow in narrow blood vessels. As a result, red blood cells often accumulate upstream of a white cell in a capillary with a single file of cells, whereas downstream of the white cell a red cell depleted region is formed. When the white cell enters a postcapillary vessel with increased diameter, the following red cells will pass the slower white cell and thereby displace it away from the vessel axis toward the wall. Then interaction between the white cell and the endothelium leads to adhesion, and the white cell starts rolling along the endothelium. We have investigated the detailed flow field in these small vessels with a large-scale hydrodynamic model of a capillary and a postcapillary vessel based on geometric and kinematic similarity. The capillary is simulated by a straight or divergent rigid axisymmetric tube, and white cells are simulated by spheres, red cells by flexible disks, and plasma by a Newtonian fluid. The experiments show that, under the proper circumstances, hydrodynamic collision of a disk and a sphere in a narrow tube results in the disks passing the sphere, leading to the displacement of the sphere toward the tube wall. The model reproduces qualitatively the in vivo flow field, and the results indicate that the attachment of white blood cells in postcapillaries is augmented as a result of hydrodynamic interaction with red cells.

In vivo measurements of “apparent viscosity” and microvessel hematocrit in the mesentery of the cat ☆

Abstract The arteriovenous (A-V) distribution of microvessel hematocrit (Hmicro) was determined throughout successive microvascular divisions in cat mesentery from in vivo measurements of optical density. In vitro correlations of optical density and tube hematocrit in small-bore glass tubes permitted the computation of in vivo values of Hmicro for luminal diameters ranging from 20 to 70 μm. For smaller-size vessels, Hmicro was determined by microocclusion and red cell counting. The results demonstrate a monotonic fall in the ratio of H micro H systemic from 0.80 in the 70-μm arterioles to a minimum of 0.21 in the immediate postcapillaries (10 μm diameter) followed by a subsequent monotonic rise to 0.95 in the 70-μm venules. Conservation of red cell flux throughout the mesenteric network was partially demonstrated upon applying previously established in vitro relationships between discharge and tube hematocrits, the resulting disparity being attributed to the rheological behavior of blood and possible A-V shunting of red cells. Simultaneous measurements of pressure drop and red cell velocity in unbranched arterioles during systemic hemodilution facilitated a comparison of in vivo and in vitro (cone-plate viscometer) apparent viscosities (η). No significant differences between the two approaches were found for arteriole diameters ranging from 24 to 47 μm in the absence of leukocyte-endothelium adhesion, with 0

Theoretical and experimental studies on viscoelastic properties of erythrocyte membrane

The deformation of a portion of erythrocyte during aspirational entry into a micropipette has been analyzed on the basis of a constant area deformation of an infinite plane membrane into a cylindrical tube. Consideration of the equilibrium of the membrane at the tip of the pipette has generated the relation between the aspirated length and the dimensionless time during deformational entry as well as during relaxation after the removal of aspiration pressure. Experimental studies on deformation and relaxation of normal human erythrocytes were performed with the use of micropipettes and a video dimension analyzer which allowed the continuous recording of the time-courses. The deformation consisted of an initial rapid phase with a membrane viscosity (range 0.6 x 10(-4) to 4 x 10(-4) dyn.s/cm) varying inversely with the degree of deformation and a later slow phase with a high membrane viscosity (mean 2.06 x 10(-2) dyn.s/cm) which was not correlated with the degree of deformation. The membrane viscosity of the recovery phase after 20 s of deformation (mean 5.44 x 10(-4) dyn.s/cm) was also independent of the degree of deformation. When determined after a short period of deformation (e.g., 2 s), however, membrane viscosity of the recovery phase became lower and agreed with that of the deformation phase. These results suggest that the rheological properties of the membrane can undergo dynamic changes depending on the extent and duration of deformation, reflecting molecular rearrangement in response to membrane strain.

The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells.

The migration of vascular endothelial cells (ECs) is critical in vascular remodeling. We showed that fluid shear stress enhanced EC migration in flow direction and called this “mechanotaxis.” To visualize the molecular dynamics of focal adhesion kinase (FAK) at focal adhesions (FAs), FAK tagged with green fluorescence protein (GFP) was expressed in ECs. Within 10 min of shear stress application, lamellipodial protrusion was induced at cell periphery in the flow direction, with the recruitment of FAK at FAs. ECs under flow migrated with polarized formation of new FAs in flow direction, and these newly formed FAs subsequently disassembled after the rear of the cell moved over them. The cells migrating under flow had a decreased number of FAs. In contrast to shear stress, serum did not significantly affect the speed of cell migration. Serum induced lamellipodia and FAK recruitment at FAs without directional preference. FAK(Y397) phosphorylation colocalized with GFP-FAK at FAs in both shear stress and serum experiments. The total level of FAK(Y397) phosphorylation after shear stress was lower than that after serum treatment, suggesting that the polarized change at cell periphery rather than the total level of FAK(Y397) phosphorylation is important for directional migration. Our results demonstrate the dynamics of FAK at FAs during the directional migration of EC in response to mechanical force, and suggest that mechanotaxis is an important mechanism controlling EC migration.

Blood Viscosity: Influence of Erythrocyte Aggregation

The addition of purified canine or bovine fibrinogen to suspensions of canine erythocytes in Ringer solution caused an increase in viscosity and the formation of aggregates of erythocytes. Both of these effects became increasingly pronounced as the fibrinogen concentration was raised, and they approached plateaus with 1 gram of fibrinogen per 100 milliliters. An increase in shear rate (or shear stress) reduced both the effect on viscosity and the aggregate size. The data suggest that fibrinogen causes an increase in blood viscosity and a departure from Newtonian behavior by interacting with erythrocytes to form cell aggregates which can be dispersed by shear stress.

Blood viscosity: influence of erythrocyte deformation.

Suspensions of canine and human erythocytes hardened with acetaldehyde differ from the suspensions of normal erythrocytes with respect to their rheological behavior. Normal erythrocytes can be packed by centrifugation so that the sediment volume is nearly 100 percent cells, but the hardened erythrocytes (RBCs) can be packed only to approximately 60 percent cells. At the same cell percentage the viscosity of the hardened RBC suspension is higher than that of the suspension of normal erythocytes. An increase in shear stress deforms the normal erythocytes and lowers the suspension viscosity, but has no influence on the viscosity of the hardened cell suspension. In blood with high cell percentages, the shear deformation of normal RBCs plays an important role in reducing viscosity and facilitating flow at high shear stresses.

Cell distribution in capillary networks

Abstract The distribution of red and white blood cells at diverging capillary branches in the rabbit ear chamber has been studied by means of high-speed microcinephotography. The experimental results are summarized in the form of a cell distribution function which is defined as the flux of cells as a function of the bulk flow into the daughter vessels. In capillaries the cell distribution function is nonlinear and strongly dependent on the eccentric position of the blood cells at the upstream entrance to the branch. If one daughter vessel carries the majority of the entering flow, all red blood cells are collected into the same channel. The cell distribution function is readily incorporated into a numerical network analysis to simulate the path velocity, pressure drop, and concentrations of red and white cells as a function of time throughout a capillary bed. The computational results show that, due to nonlinear cell distribution functions and nonuniform flow rates, the concentration of blood cells in a capillary network will be generally nonuniform. The flow fluctuations are the consequence of the particle nature of blood in capillaries without active changes in their diameters. Vasomotion would alter the hemodynamic conditions in upstream vessels and the influx of cells into the capillary network; therefore its effect would be superimposed on the flow fluctuations considered here.

Effects of Disturbed Flow On Endothelial Cells

Atherosclerotic lesions tend to localize at curvatures and branches of the arterial system, where the local flow is often disturbed and irregular (e.g., flow separation, recirculation, complex flow patterns, and nonuniform shear stress distributions). The effects of such flow conditions on cultured human umbilical vein endothelial cells (HUVECs) were studied in vitro by using a vertical-step flow channel (VSF). Detailed shear stress distributions and flow structures have been computed by using the finite volume method in a general curvilinear coordinate system. HUVECs in the reattachment areas with low shear stresses were generally rounded in shape. In contrast, the cells under higher shear stresses were significantly elongated and aligned with the flow direction, even for those in the area with reversed flow. When HUVECs were subjected to shearing in VSF, their actin stress fibers reorganized in association with the morphological changes. The rate of DNA synthesis in the vicinity of the flow reattachment area was higher than that in the laminar flow area. These in vitro experiments have provided data for the understanding of the in vivo responses of endothelial cells under complex flow environments found in regions of prevalence of atherosclerotic lesions.

Flow Characteristics of Human Erythrocytes through Polycarbonate Sieves

We used polycarbonate sieves with uniform cylindrical pores (2.4 to 6.8 microns in diameter) to filter suspensions of human erythrocytes (mean major diameter is 7.2 microns) in Eagle-albumin solution. With 6.8-micron sieves the pressure-flow curves are convexed to the pressure-axis at low pressures and become linear with high pressures. With 4.5-micron sieves, however, the pressure-flow relationship is linear throughout the range of study. In both types of sieves, flow rate is reduced progressively with increasing concentration of red blood cells (RBC) over a range of 0.5 to 95 percent. The resistance to flow of RBC suspensions is higher in 4.5-micron than in 6.8-micron pores. With filter pore diameters of 3.0 microns or more, the RBC concentration in the filtrate was 100 percent of that in the solution being filtered, but only 70 percent with 2.4-micron pores. The observed critical pore diameter for 100 percent cell transmission agrees with theoretical calculation based on the assumption that the RBC membrane is deformable but nonextensible. The importance of cell deformation in the passage of RBCs through small pores is shown by the inability of RBC hardened in acetaldehyde to pass pores with 6.8-micron diameter.