No abstract

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P(f)), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P(d)). Here, we report an accurate and instantaneous method for measuring the P(d) of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H(2)O/D(2)O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ(CARS)) for the external solution H(2)O/D(2)O exchange was 16.1 ms, whereas the intracellular H(2)O/D(2)O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ(CARS) for the intracellular H(2)O/D(2)O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P(d). The average P(d) values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10(-3) and 8.3 ± 2.6 × 10(-4) cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.

a b s t r a c tThe neurogenic gene Drosophila big brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis.

A laser-induced surface deformation (LISD) microscope is developed and applied to measurement of the dynamic relaxation responses of the plasma membrane in a living cell. A laser beam is tightly focused on an optional area of cell surface and the focused light induces microscopic deformation on the surface via radiation pressure. The LISD microscope not only allows non-contact and destruction-free measurement but provides power spectra of the surface responses depending on the frequency of the intensity of the laser beam. An optical system for the LISD is equipped via a microscope, allowing us to measure the relaxation responses in sub-cellular-sized regions of the plasma membrane. In addition, the forced oscillation caused by the radiation pressure for surface deformation extends the upper limit of the frequency range in the obtained power spectra to 106 Hz, which enables us to measure relaxation responses in local regions within the plasma membrane. From differences in power-law exponents at higher frequencies, it is realized that a cancerous cell obeys a weaker single power-law than a normal fibroblast cell. Furthermore, the power spectrum of a keratinocyte cell obeys a power-law with two exponents, indicating that alternative mechanical models to a conventional soft glassy rheology model (where single power-laws explain cells' responses below about 103 Hz) are needed for the understanding over a wider frequency range. The LISD microscope would contribute to investigation of microscopic cell rheology, which is important for clarifying the mechanisms of cell migration and tissue construction.

In the present study, myoglobin (Mb) and casein were investigated as representative proteins with α-helix-rich and random-coil structures, respectively. Conformational changes of hydrated proteins induced by gradual dehydration were monitored by vibrational circular dichroism (VCD) spectroscopy. In myoglobin and casein, representative α-helix-rich and random-coil proteins, respectively, an increase in left-handed optical activity in the amide I band was detected at the initial stage of dehydration, followed by an increase in opposite right-handed activity in both the amide I and II bands with further dehydration. Because the second step was observed with an increase in the turbidity of the proteins, it can be attributed to their aggregation. In contrast, because the increase in left-handed optical activity is induced by the conformational change of the proteins and is followed by the aggregation, it may derive from the increase in the regularity of the local structure in individual myoglobin or casein that triggers the aggregation.

The differences in the mechanical properties between the cortex and coat protein layer in a Bacillus subtilis spore were clarified using an atomic force microscope (AFM) and an originally developed laser-induced surface deformation (LISD) microscope. AFM force curve measurements show that the Young's modulus of the coat protein layer is ca. 66% lower compared with that of the cortex. It has been experimentally clarified that the cortex makes a greater contribution to the rigidity of spores than the coat protein layer. From comparisons of the LISD power spectra, it is revealed that the coat protein layer has two different viscoelastic regions, and that the cortex relatively has a higher viscous nature than the coat protein layer. Furthermore, the LISD power spectrum above 1 × 10 5 Hz in the coat protein layer suggests that the local region in the coat protein layer behaves as a more elastic body compared with the cortex.

Unlimited organ availability would represent a paradigm shift in transplantation. Longterm in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

An analytical technique to measure reactions in biological membranes was developed and applied to monitoring the hydrolysis reaction of phospholipids (dipalmitoylphosphatidylcholine, DPPC) by phospholipase A(2). The technique uses the time-resolved quasi-elastic laser scattering (TR-QELS) method to measure an oil/phospholipid monolayer/water membrane system by monitoring the change of interfacial tension under a noncontact condition and in real time. When the TR-QELS method is used with the newly developed oil/phospholipid monolayer/water membrane system, measurement of the hydrolysis reaction of phospholipids with long alkyl chains (C >or=16), which are the major components in biological membranes, becomes possible. The reaction progress is monitored by the increase of interfacial tension at the oil/water interface caused by the decrease of surface-active DPPC molecules due to the reaction. The characteristic phases, namely, lag, burst, and equilibrium, are observed. The relationship between the duration of the lag phase (the rate-limiting step of the reaction) and the concentration of calcium ion (an essential cofactor of the reaction) is also investigated. Increase of calcium ion concentration in the subphase is found to shorten the duration of the lag phase. In addition, the real-time measurement simplifies the estimation process for the reaction activation energy.