Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Aβ oligomerization

Ganglioside GM1 mediates the amyloid beta (Aβ) aggregation that is the hallmark of Alzheimer's disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Aβ, we examined the interaction between Aβ40 and supported planar lipid bilayers (SPBs) on mica and SiO₂ substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Aβ aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO₂ surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20 mol%, unique triangular regions formed on the mica surface and the rapidly formed Aβ aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Aβ40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Aβ40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Aβ-GM1 interactions.     

Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress

Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano-sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01-0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was estimated to range from several to several tens of pN.     

DNA Lesions Induced by Replication Stress Trigger Mitotic Aberration and Tetraploidy Development

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.     

Interannual variations in abundance and body size in Neocalanus copepods in the central North Pacific

As the integral components of zooplankton in the subarctic NorthPacific, the three Neocalanus species (N. cristatus, N. plumchrusand N. flemingeri) are characterized by an annual life cycleand rapid development in the surface layer during spring–summer.Patterns of interannual variation of abundance and body sizeof these Neocalanus species were analyzed using the time-seriesdata collected during the summers of 1979–1998 (20 years)at stations along the longitudinal transect line in the centralNorth Pacific, crossing five sub-areas (Alaska Current System,Subarctic Current System, Northern Transition Domain, SouthernTransition Domain and Subtropical Current System). In the southernsub-areas, quasi-decadal oscillation was observed for the 3-yearrunning mean of abundance and prosome length for copepoditestage 5 (C5) of the three Neocalanus species. Although the oscillationsignal diminished towards northern waters, it showed a positivephase during the early 1980s and 1990s and a negative phaseduring the late 1980s. In the northern waters, a biennial patternwas pronounced for anomalies of C5 prosome length for N. plumchrusand N. flemingeri, which was large in odd years and small ineven years. Significantly positive covariations among the threespecies were found for both abundance and prosome length aroundmid-latitude, where they were abundant. In the correlation analysis,these observed yearly patterns showed a statistically insignificantcorrelation with most environmental (integrated mean temperaturein surface waters, water column stability and chlorophyll aconcentration) or climatological (North Pacific Index and SouthernOscillation Index) variables. The regional difference of theoscillation signal and the synchronized covariation among thesespecies suggest that interannual variations of their abundanceand body size are mediated by common environmental force(s)with some spatial and temporal scales in the subarctic NorthPacific.     

Bayesian association mapping of multiple quantitative trait loci and its application to the analysis of genetic variation among <Emphasis Type="Italic">Oryza sativa</Emphasis> L. germplasms

One way to use a crop germplasm collection directly to map QTLs without using line-crossing experiments is the whole genome association mapping. A major problem with association mapping is the presence of population structure, which can lead to both false positives and failure to detect genuine associations (i.e., false negatives). Particularly in highly selfing species such as Asian cultivated rice, high levels of population structure are expected and therefore the efficiency of association mapping remains almost unknown. Here, we propose an approach that combines a Bayesian method for mapping multiple QTLs with a regression method that directly incorporates estimates of population structure. That is, the effects due to both multiple QTLs and population structure were included in our statistical model. We evaluated the efficiency of our approach in simulated- and real-trait analyses of a rice germplasm collection. Simulation analyses based on real marker data showed that our model could suppress both false-positive and false-negative rates and the error of estimation of genetic effects over single QTL models, indicating that our model has statistically desirable attributes over single QTL models. As real traits, we analyzed the size and shape of milled rice grains and found significant markers that may be linked to QTLs reported previously. Association mapping should have good prospects in highly selfing species such as rice if proper methods are adopted. Our approach will be useful for the whole genome association mapping of various selfing crop species.     

Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells

The endothelial cell-specific granule Weibel-Palade body releases vasoactive substances capable of modulating vascular inflammation. Although innate recognition of pathogens by Toll-like receptors (TLRs) is thought to play a crucial role in promotion of inflammatory responses, the molecular basis for early-phase responses of endothelial cells to bacterial pathogens has not fully been understood. We here report that human aortic endothelial cells respond to bacterial lipoteichoic acid (LTA) and synthetic bacterial lipopeptides, but not lipopolysaccharide or peptidoglycan, to induce Weibel-Palade body exocytosis, accompanied by release or externalization of the storage components von Willebrand factor and P-selectin. LTA could activate rapid Weibel-Palade body exocytosis through a TLR2- and MyD88-dependent mechanism without de novo protein synthesis. This process was at least mediated through MyD88-dependent phosphorylation and activation of phospholipase Cgamma. Moreover, LTA activated interleukin-1 receptor-associated kinase-1-dependent delayed exocytosis with de novo protein synthesis and phospholipase Cgamma-dependent activation of the NF-kappaB pathway. Increased TLR2 expression by transfection or interferon-gamma treatment increased TLR2-mediated Weibel-Palade body exocytosis, whereas reduced TLR2 expression under laminar flow decreased the response. Thus, we propose a novel role for TLR2 in induction of a primary proinflammatory event in aortic endothelial cells through Weibel-Palade body exocytosis, which may be an important step for linking innate recognition of bacterial pathogens to vascular inflammation.     

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.