Bidirectional multiciliated cell extrusion is controlled by Notch-driven basal extrusion and Piezo1-driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late-stage development, there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional, whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, response to the Notch signal is age dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo1, such that premature activation of Piezo1 leads to early extrusion while blocking Piezo1 leads to MCC maintenance. Distinct mechanisms for MCC loss underlie the importance of their removal during epithelial remodeling.

In Vitro Interaction of Melanoma-Derived Extracellular Vesicles with Collagen.

Extracellular vesicles are now considered as active contributors to melanoma progression through their capacity to modify the tumor microenvironment and to favor the formation of a pre-metastatic niche. These prometastatic roles of tumor-derived EVs would pass through their interaction with the extracellular matrix (ECM) and its remodeling, in turn providing a substrate favoring persistent tumor cell migration. Nevertheless, the capacity of EVs to directly interact with ECM components is still questionable. In this study, we use electron microscopy and a pull-down assay to test the capacity of sEVs, derived from different melanoma cell lines, to physically interact with collagen I. We were able to generate collagen fibrils coated with sEVs and to show that melanoma cells release subpopulations of sEVs that can differentially interact with collagen.

Novel centriolar defects underlie a primary ciliary dyskinesia phenotype in an adenylate kinase 7 deficient ciliated epithelium.

The skin of Xenopus embryos contains numerous multiciliated cells (MCCs), which collectively generate a directed fluid flow across the epithelial surface essential for distributing the overlaying mucous. MCCs develop into highly specialized cells to generate this flow, containing approximately 150 evenly spaced centrioles that give rise to motile cilia. MCC-driven fluid flow can be impaired when ciliary dysfunction occurs, resulting in primary ciliary dyskinesia (PCD) in humans. Mutations in a large number of genes (~50) have been found to be causative to PCD. Recently, studies have linked low levels of Adenylate Kinase 7 (AK7) gene expression to patients with PCD; however, the mechanism for this link remains unclear. Additionally, AK7 mutations have been linked to multiple PCD patients. Adenylate kinases modulate ATP production and consumption, with AK7 explicitly associated with motile cilia. Here we reproduce an AK7 PCD-like phenotype in Xenopus and describe the cellular consequences that occur with manipulation of AK7 levels. We show that AK7 localizes throughout the cilia in a DPY30 domain-dependent manner, suggesting a ciliary function. Additionally, we find that AK7 overexpression increases centriole number, suggesting a role in regulating centriole biogenesis. We find that in AK7-depleted embryos, cilia number, length, and beat frequency are all reduced, which in turn, significantly decreases the tissue-wide mucociliary flow. Additionally, we find a decrease in centriole number and an increase in sub-apical centrioles, implying that AK7 influences both centriole biogenesis and docking, which we propose underlie its defect in ciliogenesis. We propose that AK7 plays a role in PCD by impacting centriole biogenesis and apical docking, ultimately leading to ciliogenesis defects that impair mucociliary clearance.

Bidirectional multiciliated cell extrusion is controlled by Notch driven basal extrusion and Piezo 1 driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late stage development there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, receptivity to the Notch signal is age-dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo 1 such that premature activation of Piezo 1 leads to early extrusion while blocking Piezo 1 leads to MCC maintenance. Distinct mechansms for MCC loss underlie the importance of their removal during epithelial remodeling. Summay Statement: Cell extrusion typically occurs unidirectionally. We have identified a single population of multiciliated cells that extrudes bidirectionally: Notch-driven basal extrusion and Piezo 1-mediated apical extrusion.

Clathrin-coated structures support 3D directed migration through local force transmission.

Migrating cells navigate in complex environments through sensing and interpreting biochemical and/or mechanical cues. Here, we report that recently identified tubular clathrin/AP-2 lattices (TCALs), a subset of clathrin-coated structures (CCSs) that pinch collagen fibers, mechanically control directed migration along fibers decorated with ligands of CCS cargoes in three-dimensional (3D) environments. We observed that epidermal growth factor or low-density lipoprotein bound to collagen fibers leads to increased local nucleation and accumulation of TCALs. By using engineered, mixed collagen networks, we demonstrate that this mechanism selectively increases local forces applied on ligand-decorated fibers. We show that these effects depend on the ligand's receptors but do not rely on their ability to trigger signaling events. We propose that the preferential accumulation of TCALs along ligand-decorated fibers steers migration in 3D environments. We conclude that ligand-regulated, local TCAL accumulation results in asymmetric force distribution that orients cell migration in 3D environments.

Tubular clathrin/AP-2 lattices pinch collagen fibers to support 3D cell migration.

Migrating cells often use focal adhesions in order to move. Focal adhesions are less prominent in cells migrating in three-dimensional (3D) as compared with 2D environments. We looked for alternative adhesion structures supporting cell migration. We analyzed the dynamics of clathrin-coated pits in cells migrating in a 3D environment of collagen fibers. Both topological cues and local engagement of integrins triggered the accumulation of clathrin-coated structures on fibers. Clathrin/adaptor protein 2 (AP-2) lattices pinched collagen fibers by adopting a tube-like morphology and regulated adhesion to fibers in an endocytosis-independent manner. During migration, tubular clathrin/AP-2 lattices stabilized cellular protrusions by providing anchoring points to collagen fibers. Thus, tubular clathrin/AP-2 lattices promote cell adhesion that, in coordination with focal adhesions, supports cell migration in 3D.

αTAT1 controls longitudinal spreading of acetylation marks from open microtubules extremities.

Acetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time. Bias for MTs extremities was even more pronounced when using non-dynamic MTs extracted from HeLa cells. In contrast, K40 acetylation was mostly uniform along the length of MTs reconstituted from purified tubulin in vitro. Quantitative modelling of luminal diffusion of αTAT1 suggested that the uniform acetylation pattern observed in vitro is consistent with defects in the MT lattice providing lateral access to the lumen. Indeed, we observed that in vitro MTs are permeable to macromolecules along their shaft while cellular MTs are not. Our results demonstrate αTAT1 enters the lumen from open extremities and spreads K40 acetylation marks longitudinally along cellular MTs. This mode of tip-directed microtubule acetylation may allow for selective acetylation of subsets of microtubules.