Exchangeable Colloidal AFM Probes for the Quantification of Irreversible and Long-Term Interactions

An original method is presented to study single-colloid interaction with a substrate in liquid environment. Colloids, either in solution or adsorbed on a surface, are fixed by suction against the aperture of a microchanneled atomic force microscopy cantilever. Their adhesion to the substrate is measured, followed by their release via a short overpressure surge. Such colloid exchange procedure allows for 1), the quick variation of differently functionalized colloids within the same experiment; 2), the investigation of long-term interactions by leaving the colloids on a surface for a defined time before detaching them; and 3), the inspection of irreversible interactions. After validation of the method by reproducing literature results obtained with traditional colloidal atomic force microscopy, the serial use of colloids with different surface functionalization was shown on a micropatterned surface. Finally, concanavalin A-coated colloids were allowed to adsorb on human embryonic kidney cells and then detached one by one. The adhesion between cells and colloids was up to 60 nN, whereas individual cells adhered with 20 nN to the glass substrate. A cellular elastic modulus of 0.8 kPa was determined using the attached colloid as indenter.     

Displacement of the mitotic apparatus which induces ectopic polar body formation or parthenogenetic cleavage in starfish oocytes.

When the mitotic apparatus (MA) at meiosis I and II in starfish oocytes was detached from the animal pole and translocated to the other cortex, MA induced polar body formation, which indicates reattachment of MA to the cortex. MA attachment was so strong that MA at meiosis II was frequently broken into two parts during detachment and from the remnant part remaining at the cortex an aster derived and a nucleus derived from the detached part. When they were apart until the cleavage stage, the oocyte divided into the aster-containing and nucleus-containing blastomeres and, further, only the former blastomere divided repeatedly. This result indicates that the centrosome in the peripheral aster, which presumes to be discarded into the second polar body, always has the capacity of duplication but the centrosome in the inner aster, which stays in the oocyte interior, has not the capacity and confirms our previous report ( Saiki and Hamaguchi (1998) Dev. Biol. 203, 62-74). Furthermore, it is found by observing meiotic MA formation that this peculiar centrosome delivery at meiosis II is ensured by the fact that the attachment of the aster staying in the oocyte interior to the cortex occurs earlier than centrosome duplication.     

An optimized potential function for the calculation of nucleic acid interaction energies I. Base stacking

An optimized potential function for base-stacking interaction is constructed. Stacking energies between the complementary pairs of a dimer are calculated as a function of the rotational angle and separation distance. Using several different sets of atomic charges, the electrostatic component in the monopole-monopole approximation (MMA) is compared to the more refined segmented multipole–multipole representation (SMMA); the general features of the stacking minima are found to be correctly reproduced with IEHT or CNDO atomic charges. The electrostatic component is observed to control the location of stacking minima. The MMA, in general, is not a reliable approximation of the SMMA in regions away from minima; however, the MMA is reliable in predicting the location and nature of stacking minima. The attractive part of the Lennard-Jones 6–12 potential is compared to and parameterized against the expression for the second-order interaction terms composed of multipole-bond polarizability for the polarization energy and transition-dipole bond polarizabilities for approximation of the dispersion energy. The repulsive part of the Lennard-Jones potential is compared to a Kitaygorodski-type repulsive function; changing the exponent from its usual value of 12 to 11.7 gives significantly better agreement with the more refined repulsive function. Stacking minima calculated with the optimized potential method are compared with various perturbation-type treatments. The optimized potential method yields results that compare as well with melting data as do any of the more recent and expensive perturbation methods.     

Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment

Breast tumor cells enter the bloodstream long before the development of clinically evident metastasis. However, the early presence of such bloodborne cells predicts poor patient outcome. Nearly 90% of human breast tumors arise as carcinomas from mammary epithelial cells, so it is important to study how these cells respond to the detached conditions that they would experience in the bloodstream. We report here that mammary epithelial cell lines produce long and dynamic protrusions of the plasma membrane when detached. Although human and mouse mammary epithelial cell lines die by apoptosis within 16 h of detachment, this protrusive response persists for days in cells overexpressing either Bcl-2 or Bcl-xL. Unlike actin-dependent invadopodia and podosomes, these protrusions are actually enhanced by actin depolymerization with Cytochalasin-D or Latrunculin-A. Immunofluorescence and Western blotting demonstrate that the protrusions are enriched in detyrosinated Glu-tubulin, a post-translationally modified form of alpha-tubulin that is found in stabilized microtubules. Video microscopy indicates that these protrusions promote cell-cell attachment, and inhibiting microtubule-based protrusions correlates with reduced extracellular matrix attachment. Since bloodborne metastasis depends on both cell-cell and cell-matrix attachment, microtubule-based protrusions in detached mammary epithelial cells provide a novel mechanism that could influence the metastatic spread of breast tumors.     

Direct measurements of multiple adhesive alignments and unbinding trajectories between cadherin extracellular domains

Direct measurements of the interactions between antiparallel, oriented monolayers of the complete extracellular region of C-cadherin demonstrate that, rather than binding in a single unique orientation, the cadherins adhere in three distinct alignments. The strongest adhesion is observed when the opposing extracellular fragments are completely interdigitated. A second adhesive alignment forms when the interdigitated proteins separate by 70 +/- 10 A. A third complex forms at a bilayer separation commensurate with the approximate overlap of cadherin extracellular domains 1 and 2 (CEC1-2). The locations of the energy minima are independent of both the surface density of bound cadherin and the stiffness of the force transducer. Using surface element integration, we show that two flat surfaces that interact through an oscillatory potential will exhibit discrete minima at the same locations in the force profile measured between hemicylinders covered with identical materials. The measured interaction profiles, therefore, reflect the relative separations at which the antiparallel proteins adhere, and are unaffected by the curvature of the underlying substrate. The successive formation and rupture of multiple protein contacts during detachment can explain the observed sluggish unbinding of cadherin monolayers. Velocity-distance profiles, obtained by quantitative video analysis of the unbinding trajectory, exhibit three velocity regimes, the transitions between which coincide with the positions of the adhesive minima. These findings suggest that cadherins undergo multiple stage unbinding, which may function to impede adhesive failure under force.     

Adaptive timing of detachment in a tick parasitizing hole-nesting birds

In non-permanent parasites with low intrinsic mobility such as ticks, dispersal is highly dependent on host movements as well as the timing of separation from the hosts. Optimal detachment behaviour is all the more crucial in nidicolous ticks as the risk of detaching in non-suitable habitat is high. In this study, we experimentally investigated the detachment behaviour of Ixodes arboricola, a nidicolous tick that primarily infests birds roosting in tree-holes. We infested great tits with I. arboricola larvae or nymphs, and submitted the birds to 2 experimental treatments, a control treatment in which birds had normal access to nest boxes and an experimental treatment, in which the birds were prevented access to their nest boxes for varying lengths of time. In the control group, most ticks detached within 5 days, whereas in the experimental group, ticks remained on the bird for as long as the bird was prevented access (up to 14 days). This prolonged attachment caused a decrease in survival and engorgement weight in nymphs, but not in larvae. The capacity of I. arboricola larvae to extend the duration of attachment in non-suitable environments with no apparent costs, may be an adaptation to unpredictable use of cavities by roosting hosts during winter, and at the same time may facilitate dispersal of the larval instars.     

Some physicochemical factors relevant to cellular interactions

Colloidal stability theory is discussed to accomodate the conditions imposed by biological systems. It is shown that to obtain potential curves with secondary minima, Hamaker's constant must be in the range of 1–5 × 10^?14 ergs. The effect of increasing the dielectric constant is shown in theory to lower the surface potential and electrophoretic mobility but to increase the total energy of interaction. Calculations made from the theory predict the forces between model cells to be ca. 4.0 × 10^?7 dynes. By cone-plate shearing of cell aggregates, the most successful of several techniques tried and discussed, at shear rates approaching 1 × 10^?4 second^?1 (1.5 × 10^?4 dynes) semi-complete disaggregation was achieved although cell disruption was apparent; analysis of blood viscosity data indicates 5–10 × 10^?7 dynes are required to separate red cells suspended in plasma. Colloidal stability theory, while not applicable to cell systems associated by special areas of attachment, seems to describe the physicochemical interaction of freely moving or reversibly adherent cells.     

Reconsidering detached concern: the case of intensive-care nurses

The concept of detached concern, as proposed by Renée Fox in Experiment Perilous (1959), is often used in the literature today in a way she did not intend. Rather than viewing detachment and concern as dualities, scholars frequently conceive of them as dichotomous, emphasizing detachment over concern. We reconsider detached concern here through the stories 37 intensive-care nurses told about their most memorable patients. While many described efforts to keep emotionally distant from patients, they also expressed concern for patients they felt connected to, especially those who were a first for them, who were long-term primary patients, who surprised them, or who died. The care nurses provide for these patients is shaped sociologically by their training and institutional contexts and is not an aberration or indicative of their losing control of their feelings. Instead, it is evidence of the dual nature of detached concern and of the importance of viewing the concept as describing more than emotional detachment.     

Minimum Membrane Bending Energies of Fusion Pores

Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a more realistic model treating a bilayer as two monolayers. For the two-monolayer model, fusion pores were found to have metastable states with energy minima at particular values of the pore diameter and bilayer separation. Fusion pore energies were relatively insensitive to membrane thickness but highly sensitive to spontaneous curvature and membrane asymmetry. With symmetrical bilayers and monolayer spontaneous curvatures of ?0.1 nm^?1 (a typical value) separated by 6 nm (closest distance determined by repulsive hydration forces), fusion pore formation required 43–65 kT. The pore radius of ~2.25 nm fell within the range estimated from conductance measurements. With bilayer separation >6 nm, fusion pore formation required less energy, suggesting that protein scaffolds can promote fusion by bending membranes toward one another. With nonzero spontaneous monolayer curvature, the shape that minimized the energy change during fusion pore formation differed from the shape that minimized its energy after it formed. Thus, a nascent fusion pore will relax spontaneously to a new shape, consistent with the experimentally observed expansion of nascent fusion pores during viral fusion.     

Effect of Detachment on the Palatability of two Kelp Species

Many species of macroalgae survive after becoming dislodged from their primary substratum, but little is known about their capacity to express anti-herbivore defences after detachment. We examined the effect of detachment on the relative palatability of the two kelp species Lessonia nigrescens and Macrocystis integrifolia to mesograzers. Laboratory and field experiments were conducted on the northern-central coast of Chile to investigate whether (i) time after detachment and (ii) grazing on detached and attached algae could trigger internal defence mechanisms in the algae, which may have acted as deterrents to grazing. In order to examine palatability, feeding assays were run after each experiment using fresh algal pieces and artificial food. Time after detachment had a significant influence on palatability of L. nigrescens but not of M. integrifolia. During the first 12 days of detachment, detached L. nigrescens held in grazer-free laboratory tanks were not significantly more palatable than attached conspecifics from the field but thereafter detached individuals became more palatable. Floating individuals of M. integrifolia showed no effect of detachment, indicating that this alga maintains its defence after detachment. An experiment conducted in the field confirmed these results for M. integrifolia. An additional laboratory experiment confirmed that attachment status plays an important role on algal defence reaction for L. nigrescens when exposed to grazers. Detached and previously grazed individuals of this species were less palatable than grazer-free control algae, but grazing had no effect on palatability of attached algae. Our results indicate that kelps have varying capacities for development of anti-grazing responses once they become detached, possibly depending on their capacity to float and survive after detachment.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Effect of cadmium and zinc on attachment and detachment interactions of Pseudomonas fluorescens H2 with glass.

The physiological and physicochemical bases for the effect of 5, 10, 50, or 100 micrograms of Cd and Zn ml-1 on the attachment and detachment interactions of Pseudomonas fluorescens H2 with glass substrata were determined. Attachment and detachment varied with the type and concentration of metal and the time at which cells were exposed to the metal. The largely inhibitory effect of the metals on bacterial motility and physiological activity did not directly influence attachment. The amount of Cd or Zn accumulated by the cells increased with metal concentration and was greater for free than for attached cells. The hydrophobicity and negative and positive charges of the bacterial surfaces (measured by hydrophobic and electrostatic interaction chromatography) were increased by cell exposure to the metals, particularly after Cd treatment. Cells exposed to Cd prior to attachment showed increased adhesion. Zinc-treated cells did not. There was a positive correlation between adhesion and Cd concentration in the attachment solution. No such relationship existed for Zn. P. fluorescens H2 exposed to Cd prior to attachment desorbed similarly to untreated controls. Zinc pretreatment resulted in decreased desorption. Cells attached in 5 or 10 micrograms of Cd or Zn ml-1 detached less than those attached in 50 or 100 micrograms of Cd or Zn ml-1. The presence of Cd or Zn during detachment had little effect on desorption. The dominant influence of Cd and Zn on attachment and detachment appears to be through modification of the bacterial surface. In natural ecosystems, heavy metals may influence the distribution of bacteria between the solid and liquid phases.     

Differential detachment from resting hosts of replete larval and nymphal Ixodes ticks

To determine whether replete subadult Ixodes ticks detach more frequently from resting than from active hosts, diverse rodents and lizards were caged in an apparatus designed to record the ticks' sites of detachment relative to the resting site of the host. Replete larval Ixodes ricinus and Ixodes dammini accumulated mainly beneath the resting places of the mice (Apodemus agrarius and Peromyscus leucopus) most frequently parasitized in nature. Although nymphal I. ricinus similarly detached where these mice rested, nymphal I. dammini detached more randomly. When lizards were used as hosts, both subadult stages of I. ricinus tended to detach away from their main resting sites; these ticks detached from squirrels more randomly. Detachment ratios for other rodent hosts, that are abundantly infested by the larvae of these ticks in nature (Apodemus flavicollis and Clethrionomys glareolus), could not be derived because nymphs generally failed to attach. Our observations are consistent with reports that both subadult stages of I. dammini, but not the adult, feed on the same kind of nest-dwelling hosts and that the host range of I. ricinus is less focused. Detachment of mouse-feeding larvae from resting mice promotes subsequent nymphal attachment to conspecific hosts, and the absence of such behavior among nymphs facilitates access of the resulting adults to deer.     

A biophysical model for interaction of cells with a surface coat (Glycocalyx). I. Electrostatic interaction profile

A model of the primary stage of cell-cell interaction is assumed, including not only the classical electrostatic and electrodynamic energies in the sense of DLVO theory but also steric interaction energy, the energy of specific and non-specific bonds and the energy due to changes of surface potential. Furthermore, in this paper, we exploit recent advances in the understanding of the structure of the cell surface (glycocalyx), assuming the fixed electrostatic charges (dissociated groups of the glycocalyx), to be space charge densities and the glycocalyx itself to be an adsorption layer.In this first part the profile of the electrostatic potential between two cells is calculated on the basis of the linear Poisson-Boltzmann equation (analytical integration) and discussed in dependence on charge densities of the glycocalyx, the separation distance between cells and the ionic strength of the suspension medium.     

Short term viability of soft tissue detached from the skeleton of reef-building corals

We have induced soft tissue detachment from the skeleton of two colonial hard corals of the Pocilloporid family, both in vivo and in vitro. A parallel was made between polyp “bail-out”, i.e. field and laboratory-observed detachment of tissue fragments alone from the skeleton, and the dissociation method used for initiation of coral primary cell cultures. The in vitro approach provided insights into the active cellular re-arrangement mechanisms underlying coral tissue detachment. Functional polyps were not regenerated. Viability of tissue isolates detached from coral skeleton was probed for their use as a model for short-term biological assays. Cell viability dropped from 70% to 30% within the first week maintenance in vitro. Short-term isolate cultures limited to 3 days are a compromise allowing attachment of coral cells, yet preserving viability at about 70% of the total coral cell population.     

Surface Energetics to Assess Microbial Adhesion onto Fluidized Chromatography Adsorbents

Cell‐to‐support interaction and cell‐to‐cell aggregation phenomena have been studied in a model system composed of intact yeast cells and agarose‐based chromatography adsorbent surfaces. Biomass components and beaded adsorbents were characterized by contact angle determinations with three diagnostic liquids and, complementarily, by zeta potential measurements. Such experimental characterization of the interacting surfaces has allowed the calculation of interfacial free energy of interaction in aqueous media vs. distance profiles. The extent of biomass adhesion was inferred from calculations performed assuming standard chromatographic conditions, but different adsorption modes. Several stationary support/mobile phase systems were considered, i.e., ion exchange, hydrophobic interaction, and pseudo‐affinity. The calculated interaction energy minima revealed marginal attraction between cells and cation exchangers or agarose‐matrix beads (U ≤ |10–20| kT) but strong attraction with anion exchangers (U ≥ |200–1000| kT). Other systems including hydrophobic interaction and chelating beads showed intermediate energy minimum values (U <$>\approx<$> |40–100| kT) for interaction with biological particles. However, the calculations also showed that working conditions in the presence of salt can promote cell aggregation apart from cell‐to‐support interaction. Predictions based on the application of the XDLVO approach were confirmed by independent experimental methods, e.g., biomass deposition experiments and laser diffraction spectroscopy. The understanding of biomass attachment onto chromatographic supports can help in alleviating process limitations normally encountered during direct (primary) sequestration of bioproducts.     

Line tension and interaction energies of membrane rafts calculated from lipid splay and tilt

Membrane domains known as rafts are rich in cholesterol and sphingolipids, and are thought to be thicker than the surrounding membrane. If so, monolayers should elastically deform so as to avoid exposure of hydrophobic surfaces to water at the raft boundary. We calculated the energy of splay and tilt deformations necessary to avoid such hydrophobic exposure. The derived value of energy per unit length, the line tension gamma, depends on the elastic moduli of the raft and the surrounding membrane; it increases quadratically with the initial difference in thickness between the raft and surround; and it is reduced by differences, either positive or negative, in spontaneous curvature between the two. For zero spontaneous curvature, gamma is approximately 1 pN for a monolayer height mismatch of approximately 0.3 nm, in agreement with experimental measurement. Our model reveals conditions that could prevent rafts from forming, and a mechanism that can cause rafts to remain small. Prevention of raft formation is based on our finding that the calculated line tension is negative if the difference in spontaneous curvature for a raft and the surround is sufficiently large: rafts cannot form if gamma < 0 unless molecular interactions (ignored in the model) are strong enough to make the total line tension positive. Control of size is based on our finding that the height profile from raft to surround does not decrease monotonically, but rather exhibits a damped, oscillatory behavior. As an important consequence, the calculated energy of interaction between rafts also oscillates as it decreases with distance of separation, creating energy barriers between closely apposed rafts. The height of the primary barrier is a complex function of the spontaneous curvatures of the raft and the surround. This barrier can kinetically stabilize the rafts against merger. Our physical theory thus quantifies conditions that allow rafts to form, and further, defines the parameters that control raft merger.     

Seasonal capacity of attached and detached vineyard roots to support grape phylloxera (Homoptera: Phylloxeridae)

Field experiments were conducted to evaluate population densities and survival, developmental rate, and fecundity of grape phylloxera, Daktulosphaira vitifoliae (Fitch), as influenced by root attachment or detachment from mature, field-grown, Vitis vinifera L. grapevines through the growing season. Experiments were performed using artificial infestations of California biotype A grape phylloxera. Thirty-day bioassays on attached- and detached-roots were repeated monthly from May to September in 1997 (cultivar 'Carignane') and April to September in 1998 (cultivar 'Thompson Seedless'). The bioassays showed that attached roots had lower population densities than detached roots in all months of both years. Densities varied by month, tending to be higher in spring than in summer. Of the population parameters studied, survival was most influenced by attachment condition, being higher on detached than on attached roots by up to 25-fold. These results imply the importance of vine-related mortality factors to grape phylloxera population density. Influence of root attachment condition on developmental rate and fecundity was not uniform across bioassay months for either year; however, in the four out of 21 assays where there was a significant difference it favored detached roots by twofold. Fruit harvest resulted in higher survival in the July assay but not for assays in August and September; however, neither developmental rate nor fecundity was affected by harvest in any ofthe assays. We conclude that mortality rather than nutritional factors are most limiting for field populations on susceptible vines. This work suggests that detachment of roots as occurs with root girdling by root pathogens may increase grape phylloxera populations on infested, susceptible vines. These results imply that excised root bioassays over-estimate grape phylloxera virulence and underestimate rootstock resistance.     

The effect of peer separation in young chimpanzees (Pan troglodytes)

Ten chimpanzees (Pan troglogytes), aged 18–24 months, housed without mothers as two dyads and two triads, were subjected to social separation. Two issues were addressed: the effects of peer separation in chimpanzees; and differential responses by subjects living in dyads compared with those living in triads. Chimpanzees that were alone during separation reacted with high levels of “protest” alternating with “despair” throughout the separation period. The continued presence of one cagemate, during separation from a third, was a strong mitigating factor. Even when the primary attachment was formed with the absent cagemate, the remaining chimpanzees clung to each other and the levels of protest and despair, when present, were low. Upon reunion, neither “detachment” nor heightened levels of clinging were conspicuous, but there was increased social interaction. The data on separation of chimpanzees are intermediate between those of humans and monkeys separated from mothers or peers. The increased social interactions during reunion, including looking, are comparable to the visual vigilance reported for humans.     

Extracellular matrix regulation of autophagy

Integrin-mediated attachment of epithelial cells to extracellular matrix (ECM) is crucial for proper growth and survival. Although detachment leads to apoptosis, termed anoikis, recent work demonstrates that ECM detachment also robustly induces autophagy, a tightly regulated lysosomal self-digestion process that actually promotes survival. Autophagy presumably protects epithelial cells from the stresses of ECM detachment, allowing them to survive provided that they reattach in a timely manner. Currently, the intracellular signals linking integrin engagement to autophagy remain unclear, but certain growth factor, energy-sensing, and stress-response pathways represent attractive candidates. Moreover, autophagy may be a previously unrecognized mechanism utilized by detached cancer cells to survive anoikis, which may facilitate tumor cell dormancy, dissemination, and metastasis.