Alternative molecular mechanisms for force transmission at adherens junctions via ß-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

Integrating bioengineering, super-resolution microscopy and mechanobiology in autophagy research: addendum to the guidelines (4th edition).

Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell. Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy.

A computational framework for testing hypotheses of the minimal mechanical requirements for cell aggregation using early annual killifish embryogenesis as a model.

Introduction: Deciphering the biological and physical requirements for the outset of multicellularity is limited to few experimental models. The early embryonic development of annual killifish represents an almost unique opportunity to investigate de novo cellular aggregation in a vertebrate model. As an adaptation to seasonal drought, annual killifish employs a unique developmental pattern in which embryogenesis occurs only after undifferentiated embryonic cells have completed epiboly and dispersed in low density on the egg surface. Therefore, the first stage of embryogenesis requires the congregation of embryonic cells at one pole of the egg to form a single aggregate that later gives rise to the embryo proper. This unique process presents an opportunity to dissect the self-organizing principles involved in early organization of embryonic stem cells. Indeed, the physical and biological processes required to form the aggregate of embryonic cells are currently unknown. Methods: Here, we developed an in silico, agent-based biophysical model that allows testing how cell-specific and environmental properties could determine the aggregation dynamics of early Killifish embryogenesis. In a forward engineering approach, we then proceeded to test two hypotheses for cell aggregation (cell-autonomous and a simple taxis model) as a proof of concept of modeling feasibility. In a first approach (cell autonomous system), we considered how intrinsic biophysical properties of the cells such as motility, polarity, density, and the interplay between cell adhesion and contact inhibition of locomotion drive cell aggregation into self-organized clusters. Second, we included guidance of cell migration through a simple taxis mechanism to resemble the activity of an organizing center found in several developmental models. Results: Our numerical simulations showed that random migration combined with low cell-cell adhesion is sufficient to maintain cells in dispersion and that aggregation can indeed arise spontaneously under a limited set of conditions, but, without environmental guidance, the dynamics and resulting structures do not recapitulate in vivo observations. Discussion: Thus, an environmental guidance cue seems to be required for correct execution of early aggregation in early killifish development. However, the nature of this cue (e.g., chemical or mechanical) can only be determined experimentally. Our model provides a predictive tool that could be used to better characterize the process and, importantly, to design informed experimental strategies.

Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism.

Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

Mechanoautophagy: Synergies Between Autophagy and Cell Mechanotransduction at Adhesive Complexes.

Cells are exposed and respond to various mechanical forces and physical cues stemming from their environment. This interaction has been seen to differentially regulate various cellular processes for maintenance of homeostasis, of which autophagy represents one of the major players. In addition, autophagy has been suggested to regulate mechanical functions of the cells including their interaction with the environment. In this minireview, we summarize the state of the art of the fascinating interplay between autophagy and the mechanotransduction machinery associated with cell adhesions, that we name ¨Mechanoautophagy¨.

Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions.

Cadherin-mediated adhesions (also known as adherens junctions) are adhesive complexes that connect neighboring cells in a tissue. While the role of the actin cytoskeleton in withstanding tension at these sites of contact is well documented, little is known about the involvement of microtubules and the associated endoplasmic reticulum (ER) network in cadherin mechanotransduction. Therefore, we investigated how the organization of ER extensions in close proximity of cadherin-mediated adhesions can affect such complexes, and vice versa. Here, we show that the extension of the ER to cadherin-mediated adhesions is tension dependent and appears to be cadherin-type specific. Furthermore, the different structural organization of the ER/microtubule network seems to affect the localization of ER-bound PTP1B at cadherin-mediated adhesions. This phosphatase is involved in the modulation of vinculin, a molecular clutch which enables differential engagement of the cadherin-catenin layer with the actomyosin cytoskeleton in response to tension. This suggests a link between structural organization of the ER/microtubule network around cadherin-specific adhesions, to control the mechanotransduction of adherens junctions by modulation of vinculin conformational state.

Mechanobiology of Autophagy: The Unexplored Side of Cancer.

Proper execution of cellular function, maintenance of cellular homeostasis and cell survival depend on functional integration of cellular processes and correct orchestration of cellular responses to stresses. Cancer transformation is a common negative consequence of mismanagement of coordinated response by the cell. In this scenario, by maintaining the balance among synthesis, degradation, and recycling of cytosolic components including proteins, lipids, and organelles the process of autophagy plays a central role. Several environmental stresses activate autophagy, among those hypoxia, DNA damage, inflammation, and metabolic challenges such as starvation. In addition to these chemical challenges, there is a requirement for cells to cope with mechanical stresses stemming from their microenvironment. Cells accomplish this task by activating an intrinsic mechanical response mediated by cytoskeleton active processes and through mechanosensitive protein complexes which interface the cells with their mechano-environment. Despite autophagy and cell mechanics being known to play crucial transforming roles during oncogenesis and malignant progression their interplay is largely overlooked. In this review, we highlight the role of physical forces in autophagy regulation and their potential implications in both physiological as well as pathological conditions. By taking a mechanical perspective, we wish to stimulate novel questions to further the investigation of the mechanical requirements of autophagy and appreciate the extent to which mechanical signals affect this process.

Palmitic acid reduces the autophagic flux in hypothalamic neurons by impairing autophagosome-lysosome fusion and endolysosomal dynamics.

High-fat diet (HFD)-induced obesity is associated with increased cancer risk. Long-term feeding with HFD increases the concentration of the saturated fatty acid palmitic acid (PA) in the hypothalamus. We previously showed that, in hypothalamic neuronal cells, exposure to PA inhibits the autophagic flux, which is the whole autophagic process from the synthesis of the autophagosomes, up to their lysosomal fusion and degradation. However, the mechanism by which PA impairs autophagy in hypothalamic neurons remains unknown. Here, we show that PA-mediated reduction of the autophagic flux is not caused by lysosomal dysfunction, as PA treatment does not impair lysosomal pH or the activity of cathepsin B.Instead, PA dysregulates autophagy by reducing autophagosome-lysosome fusion, which correlates with the swelling of endolysosomal compartments that show areduction in their dynamics. Finally, because lysosomes undergo constant dynamic regulation by the small Rab7 GTPase, we investigated the effect of PA treatment on its activity. Interestingly, we found PA treatment altered the activity of Rab7. Altogether, these results unveil the cellular process by which PA exposure impairs the autophagic flux. As impaired autophagy in hypothalamic neurons promotes obesity, and balanced autophagy is required to inhibit malignant transformation, this could affect tumor initiation, progression, and/or response to therapy of obesity-related cancers.

Avaliação do shelf life de amendoim torrado e salgado, no período de 6 meses / Evaluation of shelf life of arachis and salted peanuts in the 6-month period

O presente estudo teve como objetivo avaliar a percepção do ranço, no amendoim torrado e salgado, acondicionado com e sem atmosfera modificada, com e sem adição de antioxidante natural tocoferol (0,1 e 0,15%) e alecrim (0,1%). O produto foi analisado mensalmente, no período de seis meses, sendo avaliado intensidade de sabor e odor de ranço, crocância e preferência. Amostra preparada no mês da análise foi comparada com as demais amostras. Observou-se que os antioxidantes adicionados não interferiram significativamente no desenvolvimento do sabor e do odor de ranço. As amostras acondicionadas em embalagem sem atmosfera modificada tiveram piores resultados, quanto à preferência. A crocância foi o atributo que mais sofreu impacto com o tempo, especialmente quando comparado com o produto fabricado no mês. A embalagem é o fator que mais impactou no shelf life do produto, pois os amendoins acondicionados em embalagem transparente, sem atmosfera modificada, apresentaram sabor e odor de ranço mais evidente, bem como menor crocância e menor índice de aceitação.

This study aimed to evaluate the perception of rancid in roasted and salted peanuts, packed with and without modified atmosphere, with and without addition of natural antioxidant tocopherol (0.1 and 0.15 %) and rosemary (0.1 %). The product was analyzed monthly, within six months, and assessed intensity of flavor and odor of rancid, crispness and preference. Sample prepared in the month the analysis was compared with the other samples. It was observed that the antioxidant added should not significantly interfere with the development of the flavor and odor of rancidity. The samples packed in modified atmosphere packaging without had worse results, as to preference. The crispness was attribute that suffered the most impact over time, especially when compared with the product manufactured in the month. Packaging is the factor that most impacted the shelf life of the peanuts, because the products packaged in transparent packaging without modified atmosphere showed taste and odor most obvious rancid and less crispness and lower acceptance rate.

Avaliação do shelf life de amendoim torrado e salgado, no período de 6 meses

O presente estudo teve como objetivo avaliar a percepção do ranço, no amendoim torrado e salgado, acondicionado com e sem atmosfera modificada, com e sem adição de antioxidante natural tocoferol (0,1 e 0,15%) e alecrim (0,1%). O produto foi analisado mensalmente, no período de seis meses, sendo avaliado intensidade de sabor e odor de ranço, crocância e preferência. Amostra preparada no mês da análise foi comparada com as demais amostras. Observou-se que os antioxidantes adicionados não interferiram significativamente no desenvolvimento do sabor e do odor de ranço. As amostras acondicionadas em embalagem sem atmosfera modificada tiveram piores resultados, quanto à preferência. A crocância foi o atributo que mais sofreu impacto com o tempo, especialmente quando comparado com o produto fabricado no mês. A embalagem é o fator que mais impactou no shelf life do produto, pois os amendoins acondicionados em embalagem transparente, sem atmosfera modificada, apresentaram sabor e odor de ranço mais evidente, bem como menor crocância e menor índice de aceitação.(AU)

This study aimed to evaluate the perception of rancid in roasted and salted peanuts, packed with and without modified atmosphere, with and without addition of natural antioxidant tocopherol (0.1 and 0.15 %) and rosemary (0.1 %). The product was analyzed monthly, within six months, and assessed intensity of flavor and odor of rancid, crispness and preference. Sample prepared in the month the analysis was compared with the other samples. It was observed that the antioxidant added should not significantly interfere with the development of the flavor and odor of rancidity. The samples packed in modified atmosphere packaging without had worse results, as to preference. The crispness was attribute that suffered the most impact over time, especially when compared with the product manufactured in the month. Packaging is the factor that most impacted, the shelf life of the peanuts, because the products packaged in transparent packaging without modified atmosphere showed taste and odor most obvious rancid and less crispness and lower acceptance rate.(AU)