During epithelial tissue development, repair, and homeostasis, adherens junctions (AJs) ensure intercellular adhesion and tissue integrity while allowing for cell and tissue dynamics. Mechanical forces play critical roles in AJs’ composition and dynamics. Recent findings highlight that beyond a well-established role in reinforcing cell-cell adhesion, AJ mechanosensitivity promotes junctional remodeling and polarization, thereby regulating critical processes such as cell intercalation, division, and collective migration. Here, we provide an integrated view of mechanosensing mechanisms that regulate cell-cell contact composition, geometry, and integrity under tension and highlight pivotal roles for mechanosensitive AJ remodeling in preserving epithelial integrity and sustaining tissue dynamics.
Two types of cortical actin networks act during mitotic pseudocleavage furrowing in the Drosophila syncytium, but how they interact has remained elusive. In this issue of Developmental Cell, Zhang et al. (2018) show how these networks shape each other and propose that furrowing is driven by actin polymerization-derived pushing forces.
As the result of an intricate interplay between mechanical and biochemical cues, coordinated cell dynamics are at the basis of tissue development, homeostasis and repair. Numerous studies have addressed the interplay between these two inputs and their impact on cellular dynamics. These studies largely focus on bicellular junctions (BCJs). Recent works have illuminated that tricellular junctions (TCJs), the junctions where three cells contact, play important roles in epithelial tissues beyond their well-known structural function in preserving epithelial barrier integrity. Indeed, TJCs have recently been implicated in the regulation of collective cell migration, division orientation, cell proliferation and cell mechanical properties. More generally, the TCJ distribution aligns with the cell shape and mechanical stress orientation within the tissue, while their number encapsulates the packing topology. Importantly, known regulators of growth signalling and of cell mechanical properties are also localized at TCJs. Therefore, TCJs emerge as spatial sites to sense and integrate biochemical and mechanical inputs to guide epithelial tissue dynamics.
In epithelial tissue, new cell-cell junctions are formed upon cytokinesis. To understand junction formation during cytokinesis, we explored formation of tricellular septate junctions (TCJs) in epithelium. We found that upon midbody formation, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. The septate junction protein Discs-Large and components of the TCJ, Gliotactin and Anakonda accumulate in this 4-cell structure. Subsequently, a basal movement of the midbody parallels the detachment of the neighbouring cell membranes from the midbody, the disengagement of the daughter cells from their neighbours and the reorganisation of TCJs between the two daughter cells and their neighbouring cells. While the movement of midbody is independent of the Alix and Shrub abscission regulators, the loss of Gliotactin or Anakonda function impedes both the resolution of the connection between the daughter-neighbour cells and midbody movement. TCJ proteins therefore control an additional step of cytokinesis necessary for the disentanglement of the daughter cells from their neighbours during cytokinesis.
Mitotic spindle orientation has been linked to asymmetric cell divisions, tissue morphogenesis and homeostasis. The canonical pathway to orient the mitotic spindle is composed of the cortical recruitment factor NuMA and the molecular motor dynein, which exerts pulling forces on astral microtubules to orient the spindle. Recent work has defined a novel role for NuMA as a direct contributor to force generation. In addition, the exploration of geometrical and physical cues combined with the study of classical polarity pathways has led to deeper insights into the upstream regulation of spindle orientation. Here, we focus on how cell shape, junctions and mechanical tension act to orient spindle pulling forces in epithelia, and discuss different roles for spindle orientation in epithelia.
Genetic mutations affecting chromatin modifiers are widespread in cancers. In malignant peripheral nerve sheath tumors (MPNSTs), Polycomb repressive complex 2 (PRC2), which plays a crucial role in gene silencing, is inactivated through recurrent mutations in core subunits embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), but mutations in PRC2’s main catalytic subunit enhancer of zeste homolog 2 (EZH2) have never been found. This is in contrast to myeloid and lymphoid malignancies, which harbor frequent loss-of-function mutations in EZH2. Here, we investigated whether the absence of EZH2 mutations in MPNST is due to a PRC2-independent (i.e., noncanonical) function of the enzyme or to redundancy with EZH1. We show that, in the absence of SUZ12, EZH2 remains bound to EED but loses its interaction with all other core and accessory PRC2 subunits. Through genetic and pharmacological analyses, we unambiguously establish that EZH2 is functionally inert in this context, thereby excluding a PRC2-independent function. Instead, we show that EZH1 and EZH2 are functionally redundant in the slowly proliferating MPNST precursors. We provide evidence that the compensatory function of EZH1 is alleviated upon higher proliferation. This work reveals how context-dependent redundancies can shape tumor-type specific mutation patterns in chromatin regulators.
Oriented cell division patterns tissues by modulating cell position and fate. While cell geometry, junctions, cortical tension, and polarity are known to control division orientation, relatively little is known about how these are coordinated to ensure robust patterning. Here, we systematically characterize cell division, volume, and shape changes during mouse pre-implantation development by in toto live imaging. The analysis leads us to a model in which the apical domain competes with cell shape to determine division orientation. Two key predictions of the model are verified experimentally: when outside cells of the 16-cell embryo are released from cell shape asymmetry, the axis of division is guided by the apical domain. Conversely, orientation cues from the apical domain can be overcome by applied shape asymmetry in the 8-cell embryo. We propose that such interplay between cell shape and polarity in controlling division orientation ensures robust patterning of the blastocyst and possibly other tissues.
A UV-vis, CD, and differential pulse voltammetric study was performed on the deactivation of the activity of parallel G-quadruplex/hemin DNAzymes (G4 DNAzymes) by Pb(II). The G4 DNAzyme carries a d[TC] sequence at its 3extasciiacutex end and is stabilized by potassium(I). On addition of Pb(II), the K(I) ions in the parallel G4 are replaced by Pb(II) to keep the parallel topology. Intruded Pb(II) decrease the affinity between the topology and hemin, this leads to a decrease of DNAzyme activity for catalyzing the oxidation of 2,2extasciiacutex-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by hydrogen peroxide to form a green dye with an absorption maximum at 420 nm. The assay does not use any amplification, and has a linear response in the 0.01 to 10 muM Pb(II) concentration range and a 7.1 nM limit of detection. The method was successfully applied to the analysis of spiked water samples.
The use of RNA-sequencing technologies held a promise of improved diagnostic tools based on comprehensive transcript sets. However, mining human transcriptome data for disease biomarkers in clinical specimens are restricted by the limited power of conventional reference-based protocols relying on unique and annotated transcripts. Here, we implemented a blind reference-free computational protocol, DE-kupl, to infer yet unreferenced RNA variations from total stranded RNA-sequencing datasets of tissue origin. As a bench test, this protocol was powered for detection of RNA subsequences embedded into putative long noncoding (lnc)RNAs expressed in prostate cancer. Through filtering of 1,179 candidates, we defined 21 lncRNAs that were further validated by NanoString for robust tumor-specific expression in 144 tissue specimens. Predictive modeling yielded a restricted probe panel enabling more than 90% of true-positive detections of cancer in an independent The Cancer Genome Atlas cohort. Remarkably, this clinical signature made of only nine unannotated lncRNAs largely outperformed PCA3, the only used prostate cancer lncRNA biomarker, in detection of high-risk tumors. This modular workflow is highly sensitive and can be applied to any pathology or clinical application.
The regulation of actin dynamics is essential for various cellular processes. Former evidence suggests a correlation between the function of non-conventional myosin motors and actin dynamics. Here we investigate the contribution of myosin 1b to actin dynamics using sliding motility assays. We observe that sliding on myosin 1b immobilized or bound to a fluid bilayer enhances actin depolymerization at the barbed end, while sliding on myosin II, although 5 times faster, has no effect. This work reveals a non-conventional myosin motor as another type of depolymerase and points to its singular interactions with the actin barbed end.
Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell-substratum contacts. Overall, this study shows that a balance between cell contractility and cell-cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.
The gut microbiome is an important determinant of human health. Its composition has been shown to be influenced by multiple environmental factors and likely by host genetic variation. In the framework of the Milieu Intérieur Consortium, a total of 1000 healthy individuals of western European ancestry, with a 1:1 sex ratio and evenly stratified across five decades of life (age 20-69), were recruited. We generated 16S ribosomal RNA profiles from stool samples for 858 participants. We investigated genetic and non-genetic factors that contribute to individual differences in fecal microbiome composition.
Three studies published in Cell Reports (Hinks et al., Lamichhane et al., and Leng et al.) describe the transcriptome of human and mouse mucosal-associated invariant T (MAIT) cells after cognate and noncognate stimulation. The results confirm the variability of MAIT cell effector functions and provide evidence of a new tissue-repair gene signature expressed upon T cell receptor (TCR) stimulation.
In randomized clinical trials (RCT), the analysis is based on the intent-to-treat principle to avoid any selection bias in the constitution of groups. However, estimates of overall survival can be biased when significant crossover occurs because the separation of randomized groups is lost. To handle these switches, the inverse probability of censoring weighting (IPCW) method has been proposed; however, it is still poorly used in RCT, notably because of its complex implementation. In particular, for time-to-event outcomes, it can be difficult to format data, especially when time-dependent covariates have to be managed, with different measurement times between patients. This paper aims to present the R package ipcwswitch with some guidance for the analysis of the treatment effect on survival in a hypothetical setting where all patients would have continued to take the randomization treatment. After a brief recall of the key principles of the IPCW method, each step of the implementation is described using a toy example. The guidelines are illustrated in a case study that aimed at evaluating the benefit of therapy based on tumour molecular profiling for advanced cancers, SHIVA01.
Circulating tumor cells (CTCs) are particularly rare in non-metastatic breast cancer, and the clinical validity of CTC detection in that clinical setting was initially not well recognized. A cytological CTC detection device (CellSearch) fulfilling the CLIA requirements for analytical validity was subsequently developed and, in 2008, we reported the first study (REMAGUS02) showing that distant metastasis-free survival was shorter in early breast cancer patients with one or more CTCs. In the past 10 years, other clinical studies and meta-analyses have established CTC detection as a level-of-evidence 1 prognostic biomarker for local relapses, distant relapses, and overall survival. This review summarizes available data on CTC detection and the promises of this proliferation- and subtype-independent metastasis-associated biomarker in early breast cancer patients.
In survival analysis, time-varying covariates are covariates whose value can change during follow-up. Outcomes in medical research are frequently subject to competing risks (events precluding the occurrence of the primary outcome). We review the types of time-varying covariates and highlight the effect of their inclusion in the subdistribution hazard model. External time-dependent covariates are external to the subject, can effect the failure process, but are not otherwise involved in the failure mechanism. Internal time-varying covariates are measured on the subject, can effect the failure process directly, and may also be impacted by the failure mechanism. In the absence of competing risks, a consequence of including internal time-dependent covariates in the Cox model is that one cannot estimate the survival function or the effect of covariates on the survival function. In the presence of competing risks, the inclusion of internal time-varying covariates in a subdistribution hazard model results in the loss of the ability to estimate the cumulative incidence function (CIF) or the effect of covariates on the CIF. Furthermore, the definition of the risk set for the subdistribution hazard function can make defining internal time-varying covariates difficult or impossible. We conducted a review of the use of time-varying covariates in subdistribution hazard models in articles published in the medical literature in 2015 and in the first 5 months of 2019. Seven percent of articles published included a time-varying covariate. Several inappropriately described a time-varying covariate as having an association with the risk of the outcome.
All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative “C-circle” assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.