Cell prestress. II. Contribution of microtubules

The tensegritymodel hypothesizes that cytoskeleton-based microtubules (MTs) carrycompression as they balance a portion of cell contractile stress. Totest this hypothesis, we used traction force microscopy to measuretraction at the interface of adhering human airway smooth muscle cellsand a flexible polyacrylamide gel substrate. The prediction is that ifMTs balance a portion of contractile stress, then, upon theirdisruption, the portion of stress balanced by MTs would shift to thesubstrate, thereby causing an increase in traction. Measurements weredone first in maximally activated cells (10 µM histamine) and thenagain after MTs had been disrupted (1 µM colchicine). We found that after disruption of MTs, traction increased on average by ~13%. Because in activated cells colchicine induced neither an increase inintracellular Ca²⁺ nor an increase in myosin light chainphosphorylation as shown previously, we concluded that the observedincrease in traction was a result of load shift from MTs to thesubstrate. In addition, energy stored in the flexible substrate wascalculated as work done by traction on the deformation of thesubstrate. This result was then utilized in an energetic analysis. Weassumed that cytoskeleton-based MTs are slender elastic rods supportedlaterally by intermediate filaments and that MTs buckle as the cellcontracts. Using the post-buckling equilibrium theory of Euler struts,we found that energy stored during buckling of MTs was quantitativelyconsistent with the measured increase in substrate energy afterdisruption of MTs. This is further evidence supporting the idea thatMTs are intracellular compression-bearing elements.

     

Sensory Integration Regulating Male Courtship Behavior in Drosophila

The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior.     

The CFTR M470V gene variant as a potential modifier of COPD severity: study of Serbian population

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.     

Biomechanical imaging of cell stiffness and prestress with subcellular resolution

Knowledge of cell mechanical properties, such as elastic modulus, is essential to understanding the mechanisms by which cells carry out many integrated functions in health and disease. Cellular stiffness is regulated by the composition, structural organization, and indigenous mechanical stress (or prestress) borne by the cytoskeleton. Current methods for measuring stiffness and cytoskeletal prestress of living cells necessitate either limited spatial resolution but with high speed, or spatial maps of the entire cell at the expense of long imaging times. We have developed a novel technique, called biomechanical imaging, for generating maps of both cellular stiffness and prestress that requires less than 30 s of interrogation time, but which provides subcellular spatial resolution. The technique is based on the ability to measure tractions applied to the cell while simultaneously observing cell deformation, combined with capability to solve an elastic inverse problem to find cell stiffness and prestress distributions. We demonstrated the application of this technique by carrying out detailed mapping of the shear modulus and cytoskeletal prestress distributions of 3T3 fibroblasts, making no assumptions regarding those distributions or the correlation between them. We also showed that on the whole cell level, the average shear modulus is closely associated with the average prestress, which is consistent with the data from the literature. Data collection is a straightforward procedure that lends itself to other biochemical/biomechanical interventions. Biomechanical imaging thus offers a new tool that can be used in studies of cell biomechanics and mechanobiology where fast imaging of cell properties and prestress is desired at subcellular resolution.     

Experimental tests of the cellular tensegrity hypothesis

The tensegrity model depicts the cytoskeleton (CSK) as a prestressed network of interconnected filaments. The prestress is generated by the CSK contractile apparatus and is partly balanced by traction at the cell-substrate interface and partly by CSK internal compression elements such as microtubules (MTs). A key feature of tensegrity is that the shear modulus (G) must increase in proportion with the prestress. Here we have tested that prediction as well as the idea that compression of MTs balance a portion of the cell prestress. Airway smooth muscle cells were studied. Traction microscopy was used to calculate traction. Because traction must be balanced by the stress within the cell, the prestress could be computed. Cell G was measured by oscillatory magnetic cytometry. The prestress was modulated using graded concentrations of contracting (histamine) or relaxing (isoproterenol) agonists and by disrupting MTs by colchicine. It was found that G increased in proportion with the prestress and that compression of MTs balanced a significant, but a relatively small fraction of the prestress. Taken together, these results do not disprove other models of cell deformability, nor they prove tensegrity. However, they do support a priori predictions of tensegrity. As such, it may not be necessary to invoke more complex mechanisms to explain these central features of cell deformability.     

Effect of the cytoskeletal prestress on the mechanical impedance of cultured airway smooth muscle cells.

We investigated the effect of the cytoskeletal prestress (P) on the elastic and frictional properties of cultured human airway smooth muscle cells during oscillatory loading; P is preexisting tensile stress in the actin cytoskeleton generated by the cell contractile apparatus. We oscillated (0.1 Hz, 6 Pa peak to peak) small ferromagnetic beads bound to integrin receptors and computed the storage (elastic) modulus (G') and the loss (frictional) modulus (G") from the applied torque and the corresponding bead rotation. All measurements were done at baseline and after cells were treated with graded doses of either histamine (0.1, 1, 10 microM) or isoproterenol (0.01, 0.1, 1, 10 microM). Values for P for these concentrations were taken from a previous study (Wang et al., Am J Physiol Cell Physiol, in press). It was found that G' and G", as well as P, increased/decreased with increasing doses of histamine/isoproterenol. Both G' and G" exhibited linear dependences on P: G'(Pa) = 0.20P + 82 and G"(Pa) = 0.05P + 32. The dependence of G' on P is consistent with our previous findings and with the behavior of stress-supported structures. The dependence of G" on P is a novel finding. It could be attributed to a variety of mechanisms. Some of those mechanisms are discussed in detail. We concluded that, in addition to the central mechanisms by which stress-supported structures develop mechanical stresses, other mechanisms might need to be invoked to fully explain the observed dependence of the cell mechanical properties on the state of cell contractility.     

Unveiling the nonlinear optical response of Trictenotoma childreni longhorn beetle

The wings of some insect species are known to fluoresce under illumination by ultraviolet light. Their fluorescence properties are however, not comprehensively documented. In this article, the optical properties of one specific insect, the Trictenotoma childreni yellow longhorn beetle, were investigated using both linear and nonlinear optical (NLO) methods, including one‐ and two‐photon fluorescence and second harmonic generation (SHG). These three distinct optical signals discovered in this beetle are attributed to the presence of fluorophores embedded within the scales covering their elytra. Experimental evidence collected in this study indicates that the fluorophores are non‐centrosymmetric, a fundamental requirement for SHG. This study is the first reported optical behavior of this type in insects. We described how NLO techniques can complement other more convenient approaches to achieve a more comprehensive understanding of insect scales and integument properties.      

An experimental, non-uremic rabbit model of peritoneal dialysis

Peritoneal dialysis (PD) is a well established method of depuration in uremic patients. Standard dialysis solutions currently in use are not biocompatible with the peritoneal membrane. Studying effects of dialysate on peritoneal membrane in humans is still a challenge. There is no consensus on the ideal experimental model so far. We, therefore, wanted to develop a new experimental non-uremic rabbit model of peritoneal dialysis, which would be practical, easy to conduct, not too costly, and convenient to investigate the long-term effect of dialysis fluids. The study was done on 17 healthy Chinchilla male and female rabbits, anesthetized with Thiopental in a dose of 0.5 mg/kg body mass. A catheter, specially made from Tro-soluset (Troge Medical GMBH, Hamburg, Germany) infusion system, was then surgically inserted and tunneled from animals' abdomen to their neck. The planned experimental procedure was 4 weeks of peritoneal dialysate instillation. The presented non-uremic rabbit model of peritoneal dialysis is relatively inexpensive, does not require sophisticated technology and was well tolerated by the animals. Complications such as peritonitis, dialysis fluid leakage, constipation and catheter obstruction were negligible. This model is reproducible and can be used to analyze the effects of different dialysis solutions on the rabbit peritoneal membrane.     

Effect of surface tension on alveolar surface area.

At fixed lung volume (VL), alterations in surface tension change alveolar surface area (S) and lung recoil (PL). Wilson (26), using data from fixed lungs (1, 9), quantified the isovolume change in S with PL. We reexamined this question in fresh excised rabbit lungs, with two important differences. First, we measured fractional changes in S by using diffuse light scattering, avoiding the potential upset of the balance of tissue and surface forces during fixation. Second, we altered surface tension by ventilating the lungs with nebulized polydimethylsiloxane, with much less residual fluid compared with lavage. We found that S decreased at low and mid VL (treatment surface tension > control) by about half of Wilson's estimates and was nearly unaffected by treatment at high VL. This suggests that with increased surface tension there is 1) greater septal retraction in lungs fixed by vascular perfusion compared with unfixed lungs and 2) a greater increase in PL and less loss of S than would have been predicted.