Caveolae couple mechanical stress to integrin recycling and activation.

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active ß1-integrin content on the surface of Cav1KO fibroblasts. Florescence recovery after photobleaching analysis and endocytosis/recycling assays revealed that active ß1-integrin is mostly endocytosed through the clathrin independent carrier/glycosylphosphatidyl inositol (GPI)-enriched endocytic compartment pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven ß1-integrin activation is lower in Cav1KO mouse embryonic fibroblasts (MEFs) than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.

Cells can physically sense their immediate environment by pulling and pushing through integrins, a type of proteins which connects the inside and outside of a cell by being studded through the cellular membrane. This sensing role can only be performed when integrins are in an active state. Two main mechanisms regulate the relative amount of active integrins: one controls the activation of the proteins already at the cell surface; the other, known as recycling, impacts how many new integrins are delivered to the membrane. Both processes are affected by changes in cell membrane tension, which is itself controlled by dimples (or 'caveolae' ­ little caves in Latin) present in the cell surface. Caveolae limit acute changes in tension by taking in (pinching off the dimples) or releasing (dimples flattening) segments of the membrane. However, it is still unclear how integrins and caveolae mechanically interact to regulate the ability for a cell to read its environment. To understand this process, Lolo et al. focused on mouse cells genetically manipulated to not build caveolae on their surfaces, and which cannot properly sense mechanical changes in their surroundings. These were exposed to beads covered in an integrin-binding protein and manipulated using magnetic tweezers. The manipulation showed that mutated cells bound to the beads more strongly than non-modified cells, indicating that they had more active integrins on their surface. This change was due to both an accelerated recycling mechanism (which resulted in more integrin being brought at the surface) and an increase in integrin activation (which was triggered by a higher membrane tension). Caveolae therefore couple mechanical inputs to integrin recycling and activation. Healthy tissues rely on cells correctly sensing physical changes in their environment so they can mount an appropriate response. This ability, for example, is altered in cancerous cells which start to form tumours. The findings by Lolo et al. bring together physics and biology to provide new insights into the potential mechanisms causing such impairments.

Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients.

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.

Expression of bladder cancer-associated glycans in murine tumor cell lines.

The characterization of murine cell lines is of great importance in order to identify preclinical models that could resemble human diseases. Aberrant glycosylation includes the loss, excessive or novel expression of glycans and the appearance of truncated structures. MB49 and MB49-I are currently the only two murine cell lines available for the development of preclinical bladder cancer models. The glycans Lewis X (LeX), Sialyl lewis X (SLeX) and Sialyl Tn (STn) have previously been associated with aggressiveness, dissemination and poor prognosis in human bladder cancer, additionally N-glycolyl GM3 (NGcGM3) is a neo-antigen expressed in many types of tumors; however, to the best of our knowledge, its expression has not previously been assessed in this type of cancer. Taking into account the relevance of glycans in tumor biology and considering that they can act as targets of therapies and biomarkers, the present study evaluated the expression of LeX, SLeX, STn and NGcGM3 in MB49 and MB49-I cells, in different growth conditions such as monolayer cultures, three-dimensional multicellular spheroids and mouse heterotopic and orthotopic tumors. The expression of LeX was not detected in either cell line, whereas SLeX was expressed in monolayers, spheroids and orthotopic tumors of both cell lines. STn was only identified in MB49 monolayers and spheroids. There are no reports concerning the expression of NGcGM3 in human or murine bladder cancer. In our hands, MB49 and MB49-I expressed this ganglioside in all the growth conditions evaluated. The assessment of its expression in cancer cell lines and patient tumors is of great importance, considering the relevance of this ganglioside in tumor biology. The data obtained by the present study demonstrates that glycan expression may be substantially altered depending on the growth conditions, highlighting the importance of the characterization of murine cancer models. To the best of our knowledge, the present study is the first to examine the expression of cancer-associated glycans, in the two murine cell lines available for the development of preclinical studies in bladder cancer.