Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma

Complexity of the Tensegrity Structure for Dynamic Energy and Force Distribution of Cytoskeleton during Cell Spreading

Cytoskeleton plays important roles in intracellular force equilibrium and extracellular force transmission from/to attaching substrate through focal adhesions (FAs). Numerical simulations of intracellular force distribution to describe dynamic cell behaviors are still limited. The tensegrity structure comprises tension-supporting cables and compression-supporting struts that represent the actin filament and microtubule respectively, and has many features consistent with living cells. To simulate the dynamics of intracellular force distribution and total stored energy during cell spreading, the present study employed different complexities of the tensegrity structures by using octahedron tensegrity (OT) and cuboctahedron tensegrity (COT). The spreading was simulated by assigning specific connection nodes for radial displacement and attachment to substrate to form FAs. The traction force on each FA was estimated by summarizing the force carried in sounding cytoskeletal elements. The OT structure consisted of 24 cables and 6 struts and had limitations soon after the beginning of spreading by declining energy stored in struts indicating the abolishment of compression in microtubules. The COT structure, double the amount of cables and struts than the OT structure, provided sufficient spreading area and expressed similar features with documented cell behaviors. The traction force pointed inward on peripheral FAs in the spread out COT structure. The complex structure in COT provided further investigation of various FA number during different spreading stages. Before the middle phase of spreading (half of maximum spreading area), cell attachment with 8 FAs obtained minimized cytoskeletal energy. The maximum number of 12 FAs in the COT structure was required to achieve further spreading. The stored energy in actin filaments increased as cells spread out, while the energy stored in microtubules increased at initial spreading, peaked in middle phase, and then declined as cells reached maximum spreading. The dynamic flows of energy in struts imply that microtubules contribute to structure stabilization

On the behaviour of lung tissue under tension and compression

Lung injuries are common among those who suffer an impact or trauma. The relative severity of injuries up to physical tearing of tissue have been documented in clinical studies. However, the specific details of energy required to cause visible damage to the lung parenchyma are lacking. Furthermore, the limitations of lung tissue under simple mechanical loading are also not well documented. This study aimed to collect mechanical test data from freshly excised lung, obtained from both Sprague-Dawley rats and New Zealand White rabbits. Compression and tension tests were conducted at three different strain rates: 0.25, 2.5 and 25 min−1. This study aimed to characterise the quasi-static behaviour of the bulk tissue prior to extending to higher rates. A nonlinear viscoelastic analytical model was applied to the data to describe their behaviour. Results exhibited asymmetry in terms of differences between tension and compression. The rabbit tissue also appeared to exhibit stronger viscous behaviour than the rat tissue. As a narrow strain rate band is explored here, no conclusions are being drawn currently regarding the rate sensitivity of rat tissue. However, this study does highlight both the clear differences between the two tissue types and the important role that composition and microstructure can play in mechanical response

Motion and twisting of magnetic particles ingested by alveolar macrophages in the human lung: effect of smoking and disease

BACKGROUND: Magnetic microparticles being ingested by alveolar macrophages can be used as a monitor for intracellular phagosome motions and cytoskeletal mechanical properties. These studies can be performed in the human lung after voluntary inhalation. The influence of cigarette smoking and lung diseases on cytoskeleton dependent functions was studied. METHODS: Spherical 1.3 μm diameter ferrimagnetic iron oxide particles were inhaled by 17 healthy volunteers (40 – 65 years), 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 18 patients with chronic obstructive bronchitis (COB). The retained particles were magnetized and aligned in an external 100 mT magnetic field. All magnetized particles induce a weak magnetic field of the lung, which was detected by a sensitive SQUID (superconducting quantum interference device) sensor. Cytoskeletal reorganizations within macrophages and intracellular transport cause stochastic magnetic dipole rotations, which are reflected in a decay of the magnetic lung field, called relaxation. Directed phagosome motion was induced in a weak magnetic twisting field. The resistance of the cytoplasm to particle twisting was characterized by the viscosity and the stiffness (ratio between stress to strain) of the cytoskeleton. RESULTS: One week after particle inhalation and later macrophage motility (relaxation) and cytoskeletal stiffness was not influenced by cigarette smoking, neither in healthy subjects, nor in the patients. Patients with IPF showed in tendency a faster relaxation (p = 0.06). Particle twisting revealed a non-Newtonian viscosity with a pure viscous and a viscoelastic compartment. The viscous shear was dominant, and only 27% of the shear recoiled and reflected viscoelastic properties. In patients with IPF, the stiffness was reduced by 60% (p < 0.02). An analysis of the shear rate and stress dependence of particle twisting allows correlating the rheological compartments to cytoskeletal subunits, in which microtubules mediate the pure viscous (non-recoverable) shear and microfilaments mediate the viscoelastic (recoverable) behavior. The missing correlation between relaxation and particle twisting shows that both stochastic and directed phagosome motion reflect different cytoskeletal mechanisms. CONCLUSION: Faster relaxation and a soft cytoskeleton in patients with IPF indicate alterations in cytoskeleton dependent functions of alveolar macrophages, which may cause dysfunction's in the alveolar defense, like a slower migration, a retarded phagocytosis, a disturbed phagosome lysosome fusion and an impaired clearance

Are textbook lungs really normal? A cadaveric study on the anatomical and clinical importance of variations in the major lung fissures, and the incomplete right horizontal fissure.

INTRODUCTION: The lungs have three main fissures: the right oblique fissure (ROF), right horizontal fissure (RHF), and left oblique fissure (LOF). These can be complete, incomplete or absent; quantifying the degree of completeness of these fissures is novel. Standard textbooks often refer to the fissures as complete, but awareness of variation is essential in thoracic surgery. MATERIALS AND METHODS: Fissures in 81 pairs of cadaveric lungs were classified. Oblique fissures were measured from lung hila posteriorly to the lung hila anteriorly; and the RHF measured from the ROF to the anteromedial lung edge. The degree of completeness of fissures was expressed as a percentage of the total projected length were they to be complete. The frequency and location of accessory fissures was noted. RESULTS: LOF were complete in 66/81 (81.5%), incomplete in 13/81 (16.0%) and absent in 2/81 (2.47%); ROF were complete in 52/81 (64.2%), incomplete in 29/81 (35.8%) and never absent; RHF were more variable, complete in 18/81 (22.2%), incomplete in 54/81 (66.7%) and absent in 9/81 (11.1%). LOF and ROF were on average 97.1% and 91.6% complete, respectively, being deficient posteriorly at the lung hila. The RHF on average 69.4% complete, being deficient anteromedially. There were accessory fissures in 10 left and 19 right lungs. CONCLUSIONS: This study provides a projection of the anatomy thoracic surgeons may encounter at operation, in particular the variable RHF. This knowledge is essential for optimal outcomes in both benign and oncological procedures influenced by the fissures

Push-me-pull-you: how microtubules organize the cell interior

Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces

Cell–Matrix De-Adhesion Dynamics Reflect Contractile Mechanics

Measurement of the mechanical properties of single cells is of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this study, we show that tracking cell shape dynamics during trypsin-induced de-adhesion can serve as a simple but extremely useful tool for probing the contractility of adherent cells. When treated with trypsin, both SW13−/− epithelial cells and U373 MG glioma cells exhibit a brief lag period followed by a concerted retraction to a rounded shape. The time–response of the normalized cell area can be fit to a sigmoidal curve with two characteristic time constants that rise and fall when cells are treated with blebbistatin and nocodazole, respectively. These differences can be attributed to actomyosin-based cytoskeletal remodeling, as evidenced by the prominent buildup of stress fibers in nocodazole-treated SW13−/− cells, which are also two-fold stiffer than untreated cells. Similar results observed in U373 MG cells highlights the direct association between cell stiffness and the de-adhesion response. Faster de-adhesion is obtained with higher trypsin concentration, with nocodazole treatment further expediting the process and blebbistatin treatment blunting the response. A simple finite element model confirms that faster contraction is achieved with increased stiffness