AdamTS proteases control basement membrane heterogeneity and organ shape in Drosophila.

The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.

The F-actin bundler SWAP-70 promotes tumor metastasis.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

EMT induces characteristic changes of Rho GTPases and downstream effectors with a mitosis-specific twist.

Epithelial-mesenchymal transition (EMT) is a key cellular transformation for many physiological and pathological processes ranging from cancer over wound healing to embryogenesis. Changes in cell migration, cell morphology and cellular contractility were identified as hallmarks of EMT. These cellular properties are known to be tightly regulated by the actin cytoskeleton. EMT-induced changes of actin-cytoskeletal regulation were demonstrated by previous reports of changes of actin cortex mechanics in conjunction with modifications of cortex-associated f-actin and myosin. However, at the current state, the changes of upstream actomyosin signaling that lead to corresponding mechanical and compositional changes of the cortex are not well understood. In this work, we show in breast epithelial cancer cells MCF-7 that EMT results in characteristic changes of the cortical association of Rho-GTPases Rac1, RhoA and RhoC and downstream actin regulators cofilin, mDia1 and Arp2/3. In the light of our findings, we propose that EMT-induced changes in cortical mechanics rely on two hitherto unappreciated signaling paths-i) an interaction between Rac1 and RhoC and ii) an inhibitory effect of Arp2/3 activity on cortical association of myosin II.

TMS-induced inhibition of the left premotor cortex modulates illusory social perception.

Communicative actions from one person are used to predict another person's response. However, in some cases, these predictions can outweigh the processing of sensory information and lead to illusory social perception such as seeing two people interact, although only one is present (i.e., seeing a Bayesian ghost). We applied either inhibitory brain stimulation over the left premotor cortex (i.e., real TMS) or sham TMS. Then, participants indicated the presence or absence of a masked agent that followed a communicative or individual gesture of another agent. As expected, participants had more false alarms in the communicative (i.e., Bayesian ghosts) than individual condition in the sham TMS session and this difference between conditions vanished after real TMS. In contrast to our hypothesis, the number of false alarms increased (rather than decreased) after real TMS. These pre-registered findings confirm the significance of the premotor cortex for social action predictions and illusory social perception.

Spatio-temporal dynamics of oscillatory brain activity during the observation of actions and interactions between point-light agents.

Predicting actions from non-verbal cues and using them to optimise one's response behaviour (i.e. interpersonal predictive coding) is essential in everyday social interactions. We aimed to investigate the neural correlates of different cognitive processes evolving over time during interpersonal predictive coding. Thirty-nine participants watched two agents depicted by moving point-light stimuli while an electroencephalogram (EEG) was recorded. One well-recognizable agent performed either a 'communicative' or an 'individual' action. The second agent either was blended into a cluster of noise dots (i.e. present) or was entirely replaced by noise dots (i.e. absent), which participants had to differentiate. EEG amplitude and coherence analyses for theta, alpha and beta frequency bands revealed a dynamic pattern unfolding over time: Watching communicative actions was associated with enhanced coupling within medial anterior regions involved in social and mentalising processes and with dorsolateral prefrontal activation indicating a higher deployment of cognitive resources. Trying to detect the agent in the cluster of noise dots without having seen communicative cues was related to enhanced coupling in posterior regions for social perception and visual processing. Observing an expected outcome was modulated by motor system activation. Finally, when the agent was detected correctly, activation in posterior areas for visual processing of socially relevant features was increased. Taken together, our results demonstrate that it is crucial to consider the temporal dynamics of social interactions and of their neural correlates to better understand interpersonal predictive coding. This could lead to optimised treatment approaches for individuals with problems in social interactions.

Stiffness Measurement of Drosophila Egg Chambers by Atomic Force Microscopy.

Drosophila egg chamber development requires cellular and molecular mechanisms controlling morphogenesis. Previous research has shown that the mechanical properties of the basement membrane contribute to tissue elongation of the egg chamber. Here, we discuss how indentation with the microindenter of an atomic force microscope can be used to determine an effective stiffness value of a Drosophila egg chamber. We provide information on the preparation of egg chambers prior to the measurement, dish coating, the actual atomic force microscope measurement process, and data analysis. Furthermore, we discuss how to interpret acquired data and which mechanical components are expected to influence measured stiffness values.

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Seeing a Bayesian ghost: Sensorimotor activation leads to an illusory social perception.

Based on our prior experiences we form social expectations and anticipate another person's response. Under certain conditions, these expectations can be so strong that they lead to illusory perception of another person who is actually not there (i.e., seeing a Bayesian ghost). We used EEG to investigate the neural correlates of such illusory social perception. Our results showed that activation of the premotor cortex predicted the occurrence of the Bayesian ghost, whereas its actual appearance was later accompanied by activation in sensorimotor and adjacent parietal regions. These findings confirm that our perception of others is so strongly affected by prior expectations, in such a way they can prompt illusory social perceptions associated with activity change in brain regions relevant for action perception. They also contribute to a better understanding of social interaction in healthy individuals as well as persons with mental illnesses, which can be characterized by illusory perception and social interaction difficulties.

Skin epithelial cells change their mechanics and proliferation upon snail-mediated EMT signalling.

Skin cancer is the most commonly occurring cancer in the USA and Germany, and the fourth most common cancer worldwide. Snail-dependent epithelial-mesenchymal transition (EMT) was shown to initiate and promote skin cancer. Previous studies could show that EMT changes actin cortex regulation and cellular mechanics in epithelial cells of diverse tissue origin. However, in spite of its potentially high significance in the context of skin cancer, the effect of EMT on cellular mechanics, mitotic rounding and proliferation has not been studied in skin epithelial cells so far. In this work, we show that TGF-ß-induced partial EMT results in a transformation of the mechanical phenotype of skin epithelial cells in a cell-cycle dependent manner. Concomitantly, we looked at EMT-induced changes of cell proliferation. While EMT decreases proliferation in 2D culture, we observed an EMT-induced boost of cellular proliferation when culturing cells as mechanically confined aggregates of skin epithelial cells. This proliferation boost was accompanied by enhanced mitotic rounding and composition changes of the actin cortex. We give evidence that observed EMT-induced changes depend on the EMT-upregulated transcription factor snail. Overall, our findings indicate that EMT-induced changes of cellular mechanics might play a currently unappreciated role in EMT-induced promotion of skin tumor proliferation.

Theta:gamma phase coupling and evoked gamma activity reflect the fidelity of mental templates during memory matching in visual perception.

Top-down predictions of future events shaped by prior experience are an important control mechanism to allocate limited attentional resources more efficiently and are thought to be implemented as mental templates stored in memory. Increased evoked gamma activity and theta:gamma phase-phase coupling over parieto-occipital areas have previously been observed when mental templates meet matching visual stimuli. Here, we investigated how these signatures evolve during the formation of new mental templates and how they relate to the fidelity of such. Based on single-trial feedback, participants learned to classify target shapes as matching or mismatching with preceding cue sequences. In the end of the experiment, they were asked to freely reproduce targets as means of template fidelity. We observed fidelity-dependent increments of matching-related gamma phase locking and theta:gamma phase coupling in early visual areas around 100-200-ms poststimulus over time. Theta:gamma phase synchronization and evoked gamma activity might serve as complementary signatures of memory matching in visual perception; theta:gamma phase synchronization seemingly most important in early phases of learning and evoked gamma activity being essential for transition of mental templates into long-term memory.

EMT changes actin cortex rheology in a cell-cycle-dependent manner.

The actin cortex is a key structure for cellular mechanics and cellular migration. Accordingly, cancer cells were shown to change their actin cytoskeleton and their mechanical properties in correlation with different degrees of malignancy and metastatic potential. Epithelial-mesenchymal transition (EMT) is a cellular transformation associated with cancer progression and malignancy. To date, a detailed study of the effects of EMT on the frequency-dependent viscoelastic mechanics of the actin cortex is still lacking. In this work, we have used an established atomic force microscope-based method of cell confinement to quantify the rheology of the actin cortex of human breast, lung, and prostate epithelial cells before and after EMT in a frequency range of 0.02-2 Hz. Interestingly, we find for all cell lines opposite EMT-induced changes in interphase and mitosis; whereas the actin cortex softens upon EMT in interphase, the cortex stiffens in mitosis. Our rheological data can be accounted for by a rheological model with a characteristic timescale of slowest relaxation. In conclusion, our study discloses a consistent rheological trend induced by EMT in human cells of diverse tissue origin, reflecting major structural changes of the actin cytoskeleton upon EMT.

Protein condensates as aging Maxwell fluids.

Protein condensates are complex fluids that can change their material properties with time. However, an appropriate rheological description of these fluids remains missing. We characterize the time-dependent material properties of in vitro protein condensates using laser tweezer-based active and microbead-based passive rheology. For different proteins, the condensates behave at all ages as viscoelastic Maxwell fluids. Their viscosity strongly increases with age while their elastic modulus varies weakly. No significant differences in structure were seen by electron microscopy at early and late ages. We conclude that protein condensates can be soft glassy materials that we call Maxwell glasses with age-dependent material properties. We discuss possible advantages of glassy behavior for modulation of cellular biochemistry.

Erratum: Salt-Dependent Rheology and Surface Tension of Protein Condensates Using Optical Traps [Phys. Rev. Lett. 121, 258101 (2018)].

This corrects the article DOI: 10.1103/PhysRevLett.121.258101.

EMT-Induced Cell-Mechanical Changes Enhance Mitotic Rounding Strength.

To undergo mitosis successfully, most animal cells need to acquire a round shape to provide space for the mitotic spindle. This mitotic rounding relies on mechanical deformation of surrounding tissue and is driven by forces emanating from actomyosin contractility. Cancer cells are able to maintain successful mitosis in mechanically challenging environments such as the increasingly crowded environment of a growing tumor, thus, suggesting an enhanced ability of mitotic rounding in cancer. Here, it is shown that the epithelial-mesenchymal transition (EMT), a hallmark of cancer progression and metastasis, gives rise to cell-mechanical changes in breast epithelial cells. These changes are opposite in interphase and mitosis and correspond to an enhanced mitotic rounding strength. Furthermore, it is shown that cell-mechanical changes correlate with a strong EMT-induced change in the activity of Rho GTPases RhoA and Rac1. Accordingly, it is found that Rac1 inhibition rescues the EMT-induced cortex-mechanical phenotype. The findings hint at a new role of EMT in successful mitotic rounding and division in mechanically confined environments such as a growing tumor.

Binding Dynamics of α-Actinin-4 in Dependence of Actin Cortex Tension.

Mechanosensation of cells is an important prerequisite for cellular function, e.g., in the context of cell migration, tissue organization, and morphogenesis. An important mechanochemical transducer is the actin cytoskeleton. In fact, previous studies have shown that actin cross-linkers such as α-actinin-4 exhibit mechanosensitive properties in their binding dynamics to actin polymers. However, to date, a quantitative analysis of tension-dependent binding dynamics in live cells is lacking. Here, we present a, to our knowledge, new technique that allows us to quantitatively characterize the dependence of cross-linking lifetime of actin cross-linkers on mechanical tension in the actin cortex of live cells. We use an approach that combines parallel plate confinement of round cells, fluorescence recovery after photobleaching, and a mathematical mean-field model of cross-linker binding. We apply our approach to the actin cross-linker α-actinin-4 and show that the cross-linking time of α-actinin-4 homodimers increases approximately twofold within the cellular range of cortical mechanical tension, rendering α-actinin-4 a catch bond in physiological tension ranges.

The Poisson Ratio of the Cellular Actin Cortex Is Frequency Dependent.

Cell shape changes are vital for many physiological processes such as cell proliferation, cell migration, and morphogenesis. They emerge from an orchestrated interplay of active cellular force generation and passive cellular force response, both crucially influenced by the actin cytoskeleton. To model cellular force response and deformation, cell mechanical models commonly describe the actin cytoskeleton as a contractile isotropic incompressible material. However, in particular at slow frequencies, there is no compelling reason to assume incompressibility because the water content of the cytoskeleton may change. Here, we challenge the assumption of incompressibility by comparing computer simulations of an isotropic actin cortex with tunable Poisson ratio to measured cellular force response. Comparing simulation results and experimental data, we determine the Poisson ratio of the cortex in a frequency-dependent manner. We find that the Poisson ratio of the cortex decreases in the measured frequency regime analogous to trends reported for the Poisson ratio of glassy materials. Our results therefore indicate that actin cortex compression or dilation is possible in response to acting forces at sufficiently fast timescales. This finding has important implications for the parameterization in active gel theories that describe actin cytoskeletal dynamics.

3D Microenvironment Stiffness Regulates Tumor Spheroid Growth and Mechanics via p21 and ROCK.

The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment is revealed here. Thus, the findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments.

Does cross-frequency phase coupling of oscillatory brain activity contribute to a better understanding of visual working memory?

Nesting of fast rhythmical brain activity (gamma) into slower brain waves (theta) has frequently been suggested as a core mechanism of multi-item working memory (WM) retention. It provides a better understanding of WM capacity limitations, and, as we discuss in this review article, it can lead to applications for modulating memory capacity. However, could cross-frequency coupling of brain oscillations also constructively contribute to a better understanding of the neuronal signatures of working memory compatible with theoretical approaches that assume flexible capacity limits? Could a theta-gamma code also be considered as a neural mechanism of flexible sharing of cognitive resources between memory representations in multi-item WM? Here, we propose potential variants of theta-gamma coupling that could explain WM retention beyond a fixed memory capacity limit of a few visual items. Moreover, we suggest how to empirically test these predictions in the future.