Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.     

Dynamics of fusion pores connecting membranes of different tensions

The energetics underlying the expansion of fusion pores connecting biological or lipid bilayer membranes is elucidated. The energetics necessary to deform membranes as the pore enlarges, in some combination with the action of the fusion proteins, must determine pore growth. The dynamics of pore growth is considered for the case of two homogeneous fusing membranes under different tensions. It is rigorously shown that pore growth can be quantitatively described by treating the pore as a quasiparticle that moves in a medium with a viscosity determined by that of the membranes. Motion is subject to tension, bending, and viscous forces. Pore dynamics and lipid flow through the pore were calculated using Lagrange's equations, with dissipation caused by intra- and intermonolayer friction. These calculations show that the energy barrier that restrains pore enlargement depends only on the sum of the tensions; a difference in tension between the fusing membranes is irrelevant. In contrast, lipid flux through the fusion pore depends on the tension difference but is independent of the sum. Thus pore growth is not affected by tension-driven lipid flux from one membrane to the other. The calculations of the present study explain how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. In a similar fashion, swelling of secretory granules after fusion in biological systems could promote pore enlargement during exocytosis. The calculations also show that pore expansion can be caused by pore lengthening; lengthening may be facilitated by fusion proteins.     

The interactions of nucleic acids at elevated hydrostatic pressure

The application of elevated hydrostatic pressure on the order of a few thousand bar provides insight into the molecular forces responsible for stabilizing the conformations and non-covalent interactions of biological molecules in aqueous solution. In particular, the parameters derived from these studies have enabled researchers to glean information regarding the importance of hydration in the energetics and kinetics of these systems. This review presents data concerned with the application of hydrostatic pressure to study the thermodynamics, kinetics, and structure of nucleic acids and the interactions between nucleic acids and proteins, enzymes, and drugs. These complexes often form extremely stable non-covalent complexes in which electrostatic interactions play an important role. The sensitivity of these interactions to pressure offers a valuable experimental tool for investigating the energetics of the complexes.     

ZO-1 controls endothelial adherens junctions,cell–cell tension,angiogenesis, and barrier formation

Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell–cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin–based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin–VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin–dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell–cell tension, migration, angiogenesis, and barrier formation.     

Possible mechanisms of oxygen sensing in the pulmonary circulation.

Oxygen tension is known to control the pulmonary vascular tone. We reviewed three hypotheses that try to explain the mechanism whereby hypoxia is sensed in the lung tissue. The first hypothesis concerns the role of the oxygen binding hemoprotein cytochrome P-450. Studies using various inhibitors and activators of cytochrome P-450 show that this enzyme affects pulmonary vascular tone. The data are, however, contradictory. The second hypothesis postulates that hypoxia reduces the synthesis of vasodilator oxygen radicals in the lung. This hypothesis is quite well supported by experimental data. The third hypothesis, similarly widely documented, states that slowing of the respiratory chain and altered cellular energetics is crucial for sensing of hypoxia. In this case, however, it is not exactly clear how changes in cellular energetics are connected with vascular tone. The possibility exists that changes in both the cytochrome P-450 activity and in the rate of electrons flow in the respiratory chain may alter the amount of oxygen radicals in the cells and, similarly as in the "oxygen radicals" hypothesis, govern calcium channels through the control of the redox status of these channels.     

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.     

Fed-batch culture: Modeling and application in the study of microbiol energetics

Microbial growth in the fed-batch mode is described by a simple unstructured model. The model is found to be in good agreement with agreement with the experimental observation, except under highly transient conditions. Extensive experimental data were collected and the energetics of the bacterium Klebsiella pneumoniae is evaluated. It is shown that the fed-batch culti vation is a powerful experimental tool in the study of microbial kinetics and energetics simultaneously. Methods for determining the maintenance requirements are shown and evaluated. The maintenance coefficients determined from fed-batch data are systematically smaller than those reported for continuous culture systems. Results suggest a decrease in maintenance demands at low specific growth rates.     

Membrane tension, lipid adaptation, conformational changes, and energetics in MscL gating

This study aims to explore gating mechanisms of mechanosensitive channels in terms of membrane tension, membrane adaptation, protein conformation, and energetics. The large conductance mechanosensitive channel from Mycobacterium tuberculosis (Tb-MscL) is used as a model system; Tb-MscL acts as a safety valve by releasing small osmolytes through the channel opening under extreme hypoosmotic conditions. Based on the assumption that the channel gating involves tilting of the transmembrane (TM) helices, we have performed free energy simulations of Tb-MscL as a function of TM helix tilt angle in a dimyristoylphosphatidylcholine bilayer. Based on the change in system dimensions, TM helix tilting is shown to be essentially equivalent to applying an excess surface tension to the membrane, causing channel expansion, lipid adaptation, and membrane thinning. Such equivalence is further corroborated by the observation that the free energy cost of Tb-MscL channel expansion is comparable to the work done by the excess surface tension. Tb-MscL TM helix tilting results in an expanded water-conducting channel of an outer dimension similar to the proposed fully open MscL structure. The free energy decomposition indicates a possible expansion mechanism in which tilting and expanding of TM2 facilitates the iris-like motion of TM1, producing an expanded Tb-MscL.     

Relative stabilities of nitrenium ions derived from polycyclic aromatic amines. Relationship to mutagenicity.

The relative energetics of arylamine N-hydroxylation and N-O heterolysis (ArNH2----ArNHOH----ArNH+) for condensed systems of two, three and four rings were calculated using semiempirical AM1 molecular orbital theory. The overall thermodynamics of N-hydroxylation were almost insensitive to the structure of the amine while differences in the energetics of nitrenium ion formation varied from 0 to 35 kcal mol-1. Limited correlations between the latter and the experimental TA98 and TA100 mutagenicities of the amines are presented.     

Coalescence of membrane tethers: experiments, theory, and applications

Tethers are nanocylinders of lipid bilayer membrane, arising in situations ranging from micromanipulation experiments on synthetic vesicles to the formation of dynamic tubular networks in the Golgi apparatus. Relying on the extensive theoretical and experimental works aimed to understand the physics of individual tethers formation, we addressed the problem of the interaction between two nanotubes. By using a combination of micropipette manipulation and optical tweezers, we quantitatively studied the process of coalescence that occurred when the separation distance between both vesicle-tether junctions became smaller than a threshold length. Our experiments, which were supported by an original theoretical analysis, demonstrated that the measurements of the tether force and angle between tethers at coalescence directly yield the bending rigidity, kappa, and the membrane tension, sigma, of the vesicles. Contrary to other methods used to probe the bending rigidity of vesicles, the proposed approach permits a direct measurement of kappa without requiring any control of the membrane tension. Finally, after validation of the method and proposal of possible applications, we experimentally investigated the dynamics of the coalescence process.     

Fine structure of dicyemid mesozoans,with special reference to cell junctions

The fine structure of the dicyemid mesozoan, Dicyema acuticephalum, from Octopus vulgaris, was studied with special attention to intercellular junctional complexes between various kinds of cells. Two types of intercellular junction, namely, adherens junctions and gap junctions, were found in both vermiform stages and in infusoriform embryos. Adherens junctions were classified into two types. Zonulae adherentes-like junctions were observed between adjacent peripheral cells at vermiform stages, between adjacent external cells of infusoriform embryos, and between members of groups of internal cells that covered the urn in infusoriform embryos. Maculae adherentes-like junctions were seen between a peripheral cell and an axial cell at vermiform stages. In infusoriform embryos, these junctions were observed between various types of cells, excluding urn cells. Gap junctions were found between adjacent peripheral cells at vermiform stages, whereas in infusoriform embryos these junctions were located between various types of cells excluding urn cells. Dicyemids might be the most primitive multicellular animals to possess these basic types of cell junctions. Ciliary rootlet systems at vermiform stages and in infusoriform embryos were unique in structure compared with those of other primitive multicellular animals. J Morphol 231:297–305, 1997. © 1997 Wiley-Liss, Inc.     

The plastic cell: mechanical deformation of cells and tissues

Epithelial cells possess the ability to change their shape in response to mechanical stress by remodelling their junctions and their cytoskeleton. This property lies at the heart of tissue morphogenesis in embryos. A key feature of embryonic cell shape changes is that they result from repeated mechanical inputs that make them partially irreversible at each step. Past work on cell rheology has rarely addressed how changes can become irreversible in a complex tissue. Here, we review new and exciting findings dissecting some of the physical principles and molecular mechanisms accounting for irreversible cell shape changes. We discuss concepts of mechanical ratchets and tension thresholds required to induce permanent cell deformations akin to mechanical plasticity. Work in different systems has highlighted the importance of actin remodelling and of E-cadherin endocytosis. We also list some novel experimental approaches to fine-tune mechanical tension, using optogenetics, magnetic beads or stretching of suspended epithelial tissues. Finally, we discuss some mathematical models that have been used to describe the quantitative aspects of accounting for mechanical cell plasticity and offer perspectives on this rapidly evolving field.     

The energetics of organic synthesis inside and outside the cell

Thermodynamic modelling of organic synthesis has largely been focused on deep-sea hydrothermal systems. When seawater mixes with hydrothermal fluids, redox gradients are established that serve as potential energy sources for the formation of organic compounds and biomolecules from inorganic starting materials. This energetic drive, which varies substantially depending on the type of host rock, is present and available both for abiotic (outside the cell) and biotic (inside the cell) processes. Here, we review and interpret a library of theoretical studies that target organic synthesis energetics. The biogeochemical scenarios evaluated include those in present-day hydrothermal systems and in putative early Earth environments. It is consistently and repeatedly shown in these studies that the formation of relatively simple organic compounds and biomolecules can be energy-yielding (exergonic) at conditions that occur in hydrothermal systems. Expanding on our ability to calculate biomass synthesis energetics, we also present here a new approach for estimating the energetics of polymerization reactions, specifically those associated with polypeptide formation from the requisite amino acids.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Mechanical tension induces lateral movement of intramembrane components of the tight junction: studies on mouse mammary cells in culture

Occluding junctions of mammary epithelial cells in nonproliferating primary culture occasionally display an atypical pattern of intramembrane strands oriented predominantly perpendicular, instead of roughly parallel, to the apical border of the junction. To test whether the orienting influence was a centripetal cytoskeletal tension often observed in epithelial sheets on fixed substrates, we seeded cells at low density; this allows them to spread maximally while forming a barely confluent pavement. The result was a fourfold increase in the percentage of junctions with the strongly aligned, atypical pattern. Closely similar configurations were observed as the earliest detectable effect of chelation of extracellular Ca++, which induced pronounced centripetal contraction of the cell body. Externally imposed tension, applied so as to stretch cells in one direction only, affected the positions of strands in stretched junctions as might be predicted, by flattening their undulations, increasing their alignment parallel to the apical border. Thus mechanical tension alone, whether inherent in the cytoskeleton or imposed on the cell surface by exogenous force, can cause coordinate lateral displacement of macromolecular assemblies within the membranes of both joined cells.     

A simple model to link hemodynamics, fatigue and endurance in static work

This paper combines assumptions on blood flow changes during static work, on fatigue resulting from a shortage of chemical fuel, and on energy supply and demand considerations, to construct a systems model for endurance time in the maintenance of isometric muscle tension. All these quantitative assumptions are based on published experimental findings. The model is applied to published data on isometric endurance and a fit yielding R2 = 0.98 obtained on a composite data set.     

Energetics of Isometric and Isotonic Twitches in Toad Sartorius

Contractile energetics have been studied in twitches of toad sartorius muscle at 6-7°C. Isometric and isotonic energy production has been measured and plotted against a wide range of developed tensions and tension-time integrals. These parameters were varied by altering the isotonic load or by changing the preset isometric length. The isometric tension-independent heat was 1.12 ±0.18 (SD) mcal/g. The isometric heat coefficient Pl₀/H was 12.0 ±1.4 in muscles having twitch to tetanus ratios ranging from 0.4 to 0.6. Isometric enthalpy increased monotonically with tension or tension-time integral but the correlation between isometric heat and these parameters was poor. Isotonic enthalpy consumption was always higher than isometric enthalpy for any given tension or tension-time integral; however, isotonic heat production was consistently less than isometric heat production. The isotonic heat for the highest load (3 g) was not significantly different from the isometric tension-independent heat. Thus isotonic heat production first decreased and then increased with increasing tension or tension-time integral. In the discussion it is shown that the results conflict with all current interpretations of muscle energetics.     

Behavior, Energy and Fitness

SYNOPSIS. Fitness relations in behavioral energetics can bestudied using the optimality approach (cost-benefit analysis),correlational analysis of selection, the experimental approachand the comparative method, as well as other approaches. Theseapproaches ask different questions, have different virtues anddifferent deficiencies. By using the approaches in combinationwe could gain new understanding of the relationships betweenbehavior, energy and fitness.     

Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex

Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two-hybrid screen. Vezatin is a ubiquitous protein of adherens cell-cell junctions, where it interacts with both myosin VIIA and the cadherin-catenins complex. Its recruitment to adherens junctions implicates the C-terminal region of alpha-catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin-catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell-cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane-membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA-defective mutants, inactivity of the vezatin-myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.