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Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2−/−, integrin β1−/−, and talin1−/− cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPα−/− cells spread to the same area irrespective of rigidity on FN surfaces but spread 3× greater on rigid collagen IV-coated surfaces than soft. RPTPα and αvβ3 integrins were shown previously to be colocalized at leading edges and antibodies to αvβ3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPα−/− cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPα and αvβ3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.

Single-molecule tracking of membrane proteins has become an important tool for investigating dynamic processes in live cells, such as cell signaling, membrane compartmentation or trafficking. The extraction of relevant parameters, such as interaction times between molecular partners or confinement-zone sizes, from the trajectories of single molecules requires appropriate statistical methods. Here we report a new tool, the speed correlation index, designed to detect transient periods of directed motion within trajectories of diffusing molecules. The ability to detect such events in a wide range of biologically relevant parameter values (speed, diffusion coefficient, and durations of the directed period) was first established on simulated data. The method was next applied to analyze the trajectories of quantum-dot-labeled GABAA receptors in nerve growth cones. The use of the speed correlation index revealed that the receptors had a “conveyor belt” type of motion due to temporary interactions (∼4.0 s) between the receptors and the microtubules, leading to an average directed motion (velocity ∼0.3 μm s−1) in the growth-cone membrane. Our observations point to the possibility of a cytoskeleton-dependent redistribution of the sensing molecules in the membrane, which could play a role in the modulation of the cell response to external signals.

Previous work using glass microneedles to apply calibrated, localized force to neurons showed that tensile force is a sufficient signal for neurite initiation and elongation. However, previous studies did not examine the kinetics or probability of neurite initiation as a function of force or the rate of force application. Here we report the use of a new technique—magnetic bead force application—to systematically investigate the role of force in these phenomena with better ease of use and control over force than glass microneedles. Force-induced neurite initiation from embryonic chick forebrain neurons appeared to be a first-order random process whose rate increased with increasing force, and required the presence of peripheral microtubules. In addition, the probability of initiation was more than twofold lower for neurons exposed to rapid initial force ramps (450 pN/s) than for neurons exposed to slower ramps (1.5 and 11 pN/s). We observed a low force threshold for elongation (15–100 pN), which was not previously detected in chick forebrain neurites elongated by glass microneedles. Finally, neurites subjected to constant force elongated at variable instantaneous rates, and switched abruptly between elongation and retraction, similar to spontaneous, growth-cone-mediated outgrowth and microtubule dynamic instability.

The regulated ability of integrin αIIb β3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. We have developed a model system based on laser tweezers, enabling us to measure specific rupture forces needed to separate single receptor-ligand complexes. First of all, we performed a thorough and statistically representative analysis of nonspecific protein-protein binding versus specific αIIb β3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified αIIb β3 and fibrinogen, covalently attached to underlying surfaces, ranged from ∼20 to 150 pN. This distribution could be fit with a sum of an exponential curve for weak to moderate (20–60 pN) forces, and a Gaussian curve for strong (>60 pN) rupture forces that peaked at 80–90 pN. The interactions corresponding to these rupture force regimes differed in their susceptibility to αIIb β3 antagonists or Mn 2+, an αIIb β3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly >3.5-fold but did not affect the shape of the rupture force distribution, indicating that the measurements represent single-molecule binding. The yield strength of αIIb β3-fibrinogen interactions was independent of the loading rate (160–16,000 pN/s), whereas their binding probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of αIIb β3 and fibrinogen revealed at the single-molecule level.

The fluid dynamic interaction of cavitation bubbles with adherent cells on a substrate is experimentally investigated. We find that the nonspherical collapse of bubbles near to the boundary is responsible for cell detachment. High-speed photography reveals that a wall bounded flow leads to the detachment of cells. Cells at the edge of the circular area of detachment are found to be permanently porated, whereas cells at some distance from the detachment area undergo viable cell membrane poration (sonoporation). The wall flow field leading to cell detachment is modeled with a self-similar solution for a wall jet, together with a kinetic ansatz of adhesive bond rupture. The self-similar solution for the δ-type wall jet compares very well with the full solution of the Navier-Stokes equation for a jet of finite thickness. Apart from annular sites of sonoporation we also find more homogenous patterns of molecule delivery with no cell detachment.

Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10–10,000 Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of ≥100 Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, β-tubulin III, was observed on vmIPNs of 500 Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels (∼100–500 Pa) greatly favored neurons, whereas harder gels (∼1,000–10,000 Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of ∼10 Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.

The mobilities of transmembrane adhesion proteins are key underlying physical factors that contribute to neutrophil adhesion and arrest during inflammation. Here we present a novel (to our knowledge) fluorescence recovery after photobleaching system and a complementary analytical model to measure the mobility of the four key receptors involved in the adhesion cascade: L-selectin, PSGL-1, Mac-1, and LFA-1 for resting, spherical, and human neutrophils. In general, we find that β2 integrins (Mac-1, LFA-1) have mobilities 3–7 times faster than rolling associated molecules (L-selectin; PSGL-1), but that the mobilities within each of these groups are indistinguishable. Increasing temperature (room temperature versus 37°C) results in increased mobility, in all cases, and the use of a bivalent antibody label (mAb versus Fab) decreases mobility, except in the case of rolling associated molecules at room temperature. Disrupting the actin cytoskeleton increased mobility except that the highest mobilities measured for integrins (D = 1.2 × 10−9 cm2/s; 37°C, Fab) are not affected by actin poisons and approach the expected value for free diffusion. Although evidence of cytoskeletal hindrance of integrin mobility has been found in other systems, our data suggest such hindrance does not limit bulk integrin diffusion in resting neutrophils over distances and times important for adhesive plaque formation.

Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions.

G-proteins transduce signals along diverse pathways, but the factors involved in pathway selection are largely unknown. Here, we have studied the ability of G α q to select between two effectors—mammalian inositide-specific phospholipase C β (PLC β) and phosphoinositide-3-kinase (PI3K)—in human embryonic kidney 293 cells. These studies were carried out by measuring interactions between eCFP- and eYFP-tagged proteins using Forster resonance energy transfer in the basal state and during stimulation. Instead of association of G α q with effectors through diffusion and exchange, we found separate and stable pools of G α q-PLC β and G α q-PI3K complexes existing throughout the stimulation cycle. These separate complexes existed despite the ability of G α q to simultaneously bind both effectors as determined by in vitro measurements using purified proteins. Preformed G-protein/effector complexes will limit the number of pathways that a given signal will take, which may simplify predictive models.

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic \"recruitment signatures\" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.