Daniel P. Zitterbart

Mechano-coupling and regulation of contractility by the vinculin tail domain.

Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site.

Vinculin Facilitates Cell Invasion into Three-dimensional Collagen Matrices

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

Neutrophil morphology and migration are affected by substrate elasticity.

To reach sites of inflammation, neutrophils execute a series of adhesion and migration events that include transmigration through the vascular endothelium and chemotaxis through the vicinal extracellular matrix until contact is made with the point of injury or infection. These in vivo microenvironments differ in their mechanical properties. Using polyacrylamide gels of physiologically relevant elasticity in the range of 5 to 100 kPa and coated with fibronectin, we tested how neutrophil adhesion, spreading, and migration were affected by substrate stiffness. Neutrophils on the softest gels showed only small changes in spread area, whereas on the stiffest gels they showed a 3-fold increase. During adhesion and migration, the magnitudes of the distortions induced in the gel substrate were independent of substrate stiffness, corresponding to the generation of significantly larger traction stresses on the stiffer gels. Cells migrated more slowly but more persistently on stiffer substrates, which resulted in neutrophils moving greater distances over time despite their slower speeds. The largest tractions were localized to the posterior of migrating neutrophils and were independent of substrate stiffness. Finally, the phosphatidylinositol 3-kinase inhibitor LY294002 obviated the ability to sense substrate stiffness, suggesting that phosphatidylinositol 3-kinase plays a mechanistic role in neutrophil mechanosensing.

Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.

Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors. To analyze invasive behavior, we compared highly metastatic sarcoma cells with parental nonmetastatic cells. The metastatic cells exhibited a functional up-regulation of Rho/ROCK signaling and, similarly to carcinoma cells, an amoeboid mode of invasion. Using confocal and traction force microscopy, we showed that an up-regulation of Rho/ROCK signaling leads to increased cytoskeletal dynamics, myosin light chain localization, and increased tractions at the leading edge of the cells and that all of these contributed to increased cell invasiveness in a three-dimensional collagen matrix. We conclude that cells of mesenchymal origin can use the amoeboid nonmesenchymal mode of invasion as their primary invading mechanism and show the dependence of ROCK-mediated amoeboid mode of invasion on the increased capacity of cells to generate force. (Mol Cancer Res 2008;6(9):1410–20)

Breakdown of the Endothelial Barrier Function in Tumor Cell Transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.

Coordinated Movements Prevent Jamming in an Emperor Penguin Huddle

For Emperor penguins (Aptenodytes forsteri), huddling is the key to survival during the Antarctic winter. Penguins in a huddle are packed so tightly that individual movements become impossible, reminiscent of a jamming transition in compacted colloids. It is crucial, however, that the huddle structure is continuously reorganized to give each penguin a chance to spend sufficient time inside the huddle, compared with time spent on the periphery. Here we show that Emperor penguins move collectively in a highly coordinated manner to ensure mobility while at the same time keeping the huddle packed. Every 30–60 seconds, all penguins make small steps that travel as a wave through the entire huddle. Over time, these small movements lead to large-scale reorganization of the huddle. Our data show that the dynamics of penguin huddling is governed by intermittency and approach to kinetic arrest in striking analogy with inert non-equilibrium systems, including soft glasses and colloids.

The origin of traveling waves in an emperor penguin huddle

Emperor penguins breed during the Antarctic winter and have to endure temperatures as low as 50 C and wind speeds of up to 200kmh 1 . To conserve energy, they form densely packed huddles with a triangular lattice structure. Video recordings from previous studies revealed coordinated movements in regular wave-like patterns within these huddles. It is thought that these waves are triggered by individual penguins that locally disturb the huddle structure, and that the traveling wave serves to remove the lattice defects and restore order. The mechanisms that govern wave propagation are currently unknown, however. Moreover, it is unknown if the waves are always triggered by the same penguin in a huddle. Here, we present a model in which the observed wave patterns emerge from simple rules involving only the interactions between directly neighboring individuals, similar to the interaction rules found in other jammed systems, e.g. between cars in a traffic jam. Our model predicts that a 6 Authors to whom any correspondence should be addressed.

Automatic round-the-clock detection of whales for mitigation from underwater noise impacts.

Loud hydroacoustic sources, such as naval mid-frequency sonars or airguns for marine geophysical prospecting, have been increasingly criticized for their possible negative effects on marine mammals and were implicated in several whale stranding events. Competent authorities now regularly request the implementation of mitigation measures, including the shut-down of acoustic sources when marine mammals are sighted within a predefined exclusion zone. Commonly, ship-based marine mammal observers (MMOs) are employed to visually monitor this zone. This approach is personnel-intensive and not applicable during night time, even though most hydroacoustic activities run day and night. This study describes and evaluates an automatic, ship-based, thermographic whale detection system that continuously scans the ship’s environs for whale blows. Its performance is independent of daylight and exhibits an almost uniform, omnidirectional detection probability within a radius of 5 km. It outperforms alerted observers in terms of number of detected blows and ship-whale encounters. Our results demonstrate that thermal imaging can be used for reliable and continuous marine mammal protection.

Effects of subsampling of passive acoustic recordings on acoustic metrics

Passive acoustic monitoring is an important tool in marine mammal studies. However, logistics and finances frequently constrain the number and servicing schedules of acoustic recorders, requiring a trade-off between deployment periods and sampling continuity, i.e., the implementation of a subsampling scheme. Optimizing such schemes to each projects specific research questions is desirable. This study investigates the impact of subsampling on the accuracy of two common metrics, acoustic presence and call rate, for different vocalization patterns (regimes) of baleen whales: (1) variable vocal activity, (2) vocalizations organized in song bouts, and (3) vocal activity with diel patterns. To this end, above metrics are compared for continuous and subsampled data subject to different sampling strategies, covering duty cycles between 50% and 2%. The results show that a reduction of the duty cycle impacts negatively on the accuracy of both acoustic presence and call rate estimates. For a given duty cycle, frequent short listening periods improve accuracy of daily acoustic presence estimates over few long listening periods. Overall, subsampling effects are most pronounced for low and/or temporally clustered vocal activity. These findings illustrate the importance of informed decisions when applying subsampling strategies to passive acoustic recordings or analyses for a given target species.

The influence of sea ice, wind speed and marine mammals on Southern Ocean ambient sound

This paper describes the natural variability of ambient sound in the Southern Ocean, an acoustically pristine marine mammal habitat. Over a 3-year period, two autonomous recorders were moored along the Greenwich meridian to collect underwater passive acoustic data. Ambient sound levels were strongly affected by the annual variation of the sea-ice cover, which decouples local wind speed and sound levels during austral winter. With increasing sea-ice concentration, area and thickness, sound levels decreased while the contribution of distant sources increased. Marine mammal sounds formed a substantial part of the overall acoustic environment, comprising calls produced by Antarctic blue whales (Balaenoptera musculus intermedia), fin whales (Balaenoptera physalus), Antarctic minke whales (Balaenoptera bonaerensis) and leopard seals (Hydrurga leptonyx). The combined sound energy of a group or population vocalizing during extended periods contributed species-specific peaks to the ambient sound spectra. The temporal and spatial variation in the contribution of marine mammals to ambient sound suggests annual patterns in migration and behaviour. The Antarctic blue and fin whale contributions were loudest in austral autumn, whereas the Antarctic minke whale contribution was loudest during austral winter and repeatedly showed a diel pattern that coincided with the diel vertical migration of zooplankton.