ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.     

Multi-well plate cell contraction assay detects negatively correlated cellular responses to pharmacological inhibitors in contractility and migration

To enable large-scale screening of signaling molecules and drugs that regulate cellular contractility-associated mechanotransduction, we previously modified, particularly in terms of the capability of efficiently collecting big data, conventional methodologies using wrinkled substrates to determine the cellular contractility. Here, we present a new system to perform the wrinkle-based cell force assay in a multi-well plate format conformed to standardized geometric configurations and compatible with available technologies such as automated plate readers. With this highly improved throughput in terms of hardware as well as software using a deep learning-based technology, we evaluated the effect of treating cells with various types of pharmacological inhibitors on the cellular contractility. We found opposite responses of cells to the inhibitors between the contractility and collective migration activities. While similar inverse relationships between the contractility and migration have been reported in separate studies, our results here with the high-throughput screening system more broadly generalized these observations.     

Brain size affects the behavioural response to predators in female guppies (Poecilia reticulata)

Large brains are thought to result from selection for cognitive benefits, but how enhanced cognition leads to increased fitness remains poorly understood. One explanation is that increased cognitive ability results in improved monitoring and assessment of predator threats. Here, we use male and female guppies (Poecilia reticulata), artificially selected for large and small brain size, to provide an experimental evaluation of this hypothesis. We examined their behavioural response as singletons, pairs or shoals of four towards a model predator. Large-brained females, but not males, spent less time performing predator inspections, an inherently risky behaviour. Video analysis revealed that large-brained females were further away from the model predator when in pairs but that they habituated quickly towards the model when in shoals of four. Males stayed further away from the predator model than females but again we found no brain size effect in males. We conclude that differences in brain size affect the female predator response. Large-brained females might be able to assess risk better or need less sensory information to reach an accurate conclusion. Our results provide experimental support for the general idea that predation pressure is likely to be important for the evolution of brain size in prey species.     

Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress

Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, beta-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H(2)O(2), as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell-matrix but not cell-cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

Depth distribution of Daphnia in response to a deep-water algal maximum: the effect of body size and temperature gradient

1. In the absence of fish predation, Daphnia exploiting a deep‐water algal maximum are faced with a trade‐off. They can either dwell in the epilimnion where development in the warm water is fast, but food shortage causes low egg production, or in the hypolimnion, where food availability is high but development is slow because of low temperatures. 2. We tested the hypotheses that (i) depth distributions of various ontogenetic stages (size classes and egg‐bearing females) differ because daphnids react to light with size‐specific diel vertical migration (DVM) even in the absence of fish (residual predator avoidance hypothesis) and (ii) differently sized daphnids select different depths because the relative importance of temperature and food varies for ontogenetic stages (physiological hypothesis). We used large indoor mesocosms (Plankton Towers) to test these hypotheses experimentally. 3. Temperature was the strongest factor governing the distribution, with larger proportions of the population dwelling in the food‐rich hypolimnion if the temperature gradient was shallow. There were small but significant differences between ontogenetic stages during the day, but not at night. This suggested the existence of a ‘residual’ effect of light on depth distribution in the absence of a fish cue. 4. Although large individuals exhibited greater amplitude of DVM, the physiological hypothesis had to be rejected. A stage‐specific physiological effect is unlikely to be directly triggered by light, hence vertical movement of the individuals should not be synchronised. Rather, being forced into deeper layers by the residual light response during the day, large and egg‐bearing females experience a lower average temperature during day than juveniles. They probably compensate for this by spending longer time periods in warm waters at night.     

Expression and distribution of HSP27 in response to G418 in different human breast cancer cell lines

Heat shock proteins (HSPs) play an important role in folding, intracellular localization and degradation of cellular proteins. However, the cellular role of HSP27 is not completely understood. The conflicting results have been reported regarding stress-induced nuclear translocation of HSP27. In this study, human breast cancer cells transiently and stably expressing HSP27–EGFP chimera were utilized to observe the intracellular localization of HSP27. The data show that the transient and stable expression of HSP27–EGFP displayed distinguishingly cellular localization. The nuclear translocalization of HSP27–EGFP was correlated with the presence of G418. Experiments carried out with different human breast cancer cell lines revealed clearly different distribution patterns of endogenous HSP27. The subcellular distribution of endogenous HSP27 appeared diffuse throughout the cytoplasm in MDA435 cells. In MCF-7 and SKBR3 cells, the accumulation of the protein was distinctly seen along the cell membrane and around nucleus. Moreover, the nuclear translocation of endogenous HSP27 was stimulated by G418 only in MDA435 cells, but not in MCF-7 and SKBR3 cells. Overexpression of HSP27 has been associated with resistance to cisplatin and doxorubicin. The correlation of the expression pattern of HSP27 with the drug resistance may need to be investigated. Further studies on the intracellular function of HSP27 may take into account its interaction proteins in the cells. It may provide useful information for the identification of sensitivity of carcinoma cells to the chemotherapeutic drugs and development of more specific agents to circumvent HSP27.     

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.     

The cellular basis for phenotypic plasticity of body size in Western Spadefoot toad (Spea hammondi) tadpoles: patterns of cell growth and recruitment in response to food and temperature manipulations

Body size is highly variable within and among populations, both as a result of genetic variation and as a plastic response to environmental variation. From a proximate perspective, body size depends upon cell size, cell number, and extracellular matrix, but we know little about their independent contributions to size nor how these contributions vary with environmental influences. Here, I introduce the tail muscle of anuran tadpoles as a new system for studying this issue. Body size and tail size of tadpoles is sensitive to variation in food and temperature. I show first that tail muscle size is strongly correlated with overall body size, thus making it a good tissue to study size regulation. Second, the relative role of cell size and cell number, but not extracellular matrix, shows an interaction between food and temperature treatments and across ages. For example, in young tadpoles food effects on size are due exclusively to cell size at low temperatures but both cell size and number at high temperatures. This pattern partially reverses for older tadpoles. Despite the complexity of this interaction, the two populations compared show nearly identical patterns, suggesting that the plastic response is robust.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 499–510.