Biology subjects

Duszyc, K.

Publications and source records attributed to Duszyc, K..

9 recordsLinked to original sources

bioRxiv2026

The way less obvious: PIEZO1 supports apoptotic cell extrusion by optimizing tissue mechanical tension for homeostasis.

Apical cell extrusion is a mechanical process that allows epithelia to eliminate apoptotic cells and prevent inflammation. Mechanosensitive ion channels are often invoked for their capacity to mediate rapid mechanical responses in dynamic morphogenetic processes. Here we report an unexpected strategy for PIEZO1 to support apoptotic extrusion. PIEZO1 inhibition blocks extrusion in cultured cells and zebrafish larvae. However, although PIEZO1 mediates calcium signals during the extrusion process, we show that extrusion is instead antagonized by increase in the preexisting mechanical tension of the epithelium when PIEZO1 is disrupted. Correcting enhanced pre-stress in PIEZO1-disrupted epithelia is sufficient to rescue apoptotic extrusion, even though it does not restore dynamic calcium signals. PIEZO1 supports mechanical homeostasis through a calcium/calcineurin-dependent pathway that protects MYPT1/myosin phosphatase from degradation to limit Myosin II activation. Therefore, PIEZO1 support the morphogenetic process of apoptotic extrusion through mechanical homeostasis.

cell biology↗

bioRxiv2025

Sufficient levels of BECLIN1 are required for intestinal epithelial cell homeostasis and protection against unwanted intestinal inflammation

The prototypical autophagy regulator BECLIN1, orchestrates both autophagic and endocytic trafficking, and its homozygous deletion in the intestinal epithelium leads to intestinal disruption bearing similarities to inflammatory bowel disease (IBD). However, complete loss of BECLIN1 is rare in human disease. To model the effects of a partial reduction in BECLIN1, we examined mice with a monoallelic deletion of Becn1 in the intestinal epithelium, which decreased BECLIN1 protein levels by approximately 50%. Unlike the fatal phenotype following homozygous Becn1 deletion, the heterozygous mice were grossly normal, though they presented with significantly shorter small intestines. Gastrointestinal organoids derived from the mice also displayed disrupted endocytic trafficking and altered cytoskeletal dynamics resulting in mislocalisation of junctional proteins such as E-CADHERIN. In the mice, these changes were associated with impaired goblet cell function and maturation accompanied by abnormal mucin production, composition and secretion, resulting in compromised mucus barrier integrity. The mice also exhibited heightened susceptibility to dextran sulfate sodium (DSS)-induced colitis, emphasising the critical role of BECLIN1 in mediating protection against unregulated inflammation. Investigation of BECLIN1 levels in biopsies from patients with inflammatory bowel disease (IBD) revealed changes in its expression across distinct states of inflammation with reduced levels observed in highly inflamed regions in some patients, consistent with the gastrointestinal defects seen in mice with reduced BECLIN1. Hence, this study provides crucial insights into the multifaceted importance of BECLIN1 sufficiency in the intestinal epithelium to prevent pathophysiology. This highlights the potential of BECLIN1 as a biomarker and therapeutic target for IBD management.

cell biology↗

bioRxiv2024

BECLIN-1 is Essential for the Maintenance of Gastrointestinal Epithelial Integrity by Regulating Endocytic Trafficking, F-actin Organization and Lysosomal Function

Disrupted intestinal homeostasis and barrier function are key contributors to the development of various diseases, including inflammatory bowel disease (IBD). BECLIN-1, a core component of two class III phosphatidylinositol 3-kinase (PtdIns3K) complexes, has a dual role in autophagy and endocytic trafficking. Emerging evidence suggests it is involved in maintaining intestinal integrity which involves its endocytic trafficking function. To gain insights into the fatal gastrointestinal (GI) phenotype observed in BECLIN-1 knockout adult mice, organoids derived from these animals were used to investigate the role of BECLIN-1 in GI epithelial function. BECLIN-1 deletion led to disrupted localization of CADHERIN-1/E-CADHERIN to adherens junctions (AJs) and OCCLUDIN to tight junctions (TJs), resulting in the mislocalization of these critical barrier proteins. Lysosomal dysfunction was also observed, characterized by impaired cargo degradation. In addition, the filamentous actin (F-actin) cytoskeleton became disorganized though this was not associated with changes in the interaction between F-actin and CATENIN BETA-1/BETA-CATENIN nor in alterations in BETA-CATENIN localization, though BETA-CATENIN levels were reduced. These effects were all less pronounced or absent in organoids lacking the autophagy-only regulator ATG7 (autophagy related 7), further emphasizing the involvement of BECLIN-1s protein trafficking role in the maintenance of gut homeostasis and barrier function. These findings provide new insights into processes involved in epithelial dysfunction, deepening our understanding of mechanisms underlying intestinal disease.

cell biology↗

bioRxiv2023

Pre-existing tissue mechanical hypertension at adherens junctions disrupts apoptotic extrusion in epithelia

Apical extrusion is a tissue-intrinsic process that allows epithelia to eliminate unfit or surplus cells. This is exemplified by the early extrusion of apoptotic cells, which is critical to maintain the epithelial barrier and prevent inflammation. Apoptotic extrusion is an active mechanical process, which involves mechanotransduction between apoptotic cells and their neighbours, as well as local changes in tissue mechanics. Here we report that the pre-existing mechanical tension at adherens junctions conditions the efficacy of apoptotic extrusion. Specifically, increasing baseline mechanical tension by overexpression of a phosphomimetic Myosin II regulatory light chain (MRLC) compromises apoptotic extrusion. This occurs when tension is increased in either the apoptotic cell or its surrounding epithelium. Further, we find that the pro-inflammatory cytokine, TNF, stimulates Myosin II and increases baseline AJ tension to disrupt apical extrusion, causing apoptotic cells to be retained in monolayers. Importantly, reversal of mechanical tension with an inhibitory MRLC mutant or tropomyosin inhibitors is sufficient to restore apoptotic extrusion in TNF-treated monolayers. Together, these findings demonstrate that baseline levels of tissue tension are important determinants of apoptotic extrusion, which can potentially be co-opted by pathogenetic factors to disrupt the homeostatic response of epithelia to apoptosis.

cell biology↗

bioRxiv2023

Beclin1 is essential for intestinal homeostasis.

BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrates autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice led to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibited extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells displayed morphological abnormalities. Deletion of the autophagy regulator, ATG7, failed to elicit similar effects, suggesting novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice showed E-cadherin mislocalisation providing a mechanism linking endocytic trafficking mediated by Beclin1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has significant implications for our understanding of intestinal pathophysiology.

cell biology↗

bioRxiv2023

Desmosome-anchored intermediate filaments facilitate tension-sensitive RhoA signaling for epithelial homeostasis

Epithelia are subject to diverse forms of mechanical stress during development and post-embryonic life. They possess multiple mechanisms to preserve tissue integrity against tensile forces, which characteristically involve specialized cell-cell adhesion junctions coupled to the cytoskeleton. Desmosomes connect to intermediate filaments (IF) via desmoplakin (DP) 1,2, while the E-cadherin complex links to the actomyosin cytoskeleton in adherens junctions (AJ) 3. These distinct adhesion-cytoskeleton systems support different strategies to preserve epithelial integrity, especially against tensile stress. IFs coupled to desmosomes can passively respond to tension by strain-stiffening 4-10, whereas for AJs a variety of mechanotransduction mechanisms associated with the E-cadherin apparatus itself 11,12, or proximate to the junctions 13, can modulate the activity of its associated actomyosin cytoskeleton by cell signaling. We now report a pathway where these systems collaborate for active tension-sensing and epithelial homeostasis. We found that DP was necessary for epithelia to activate RhoA at AJ on tensile stimulation, an effect that required its capacity to couple IF to desmosomes. DP exerted this effect by facilitating the association of Myosin VI with E-cadherin, the mechanosensor for the tension-sensitive RhoA pathway at AJ 12. This connection between the DP-IF system and AJ-based tension-sensing promoted epithelial resilience when contractile tension was increased. It further facilitated epithelial homeostasis by allowing apoptotic cells to be eliminated by apical extrusion. Thus, active responses to tensile stress in epithelial monolayers reflect an integrated response of the IF- and actomyosin-based cell-cell adhesion systems.

cell biology↗

bioRxiv2023

Apical extrusion prevents apoptosis from activating an acute inflammatory program in epithelia.

Apoptosis is traditionally considered to be an immunologically silent form of cell death. Multiple mechanisms exist to ensure that apoptosis does not stimulate the immune system to cause inflammation or autoimmunity. Against this expectation, we now report epithelia are programmed to provoke, rather than suppress, inflammation in response to apoptosis. We found that an acute inflammatory response led by neutrophils occurs in zebrafish and cell culture when apoptotic epithelial cells cannot be expelled from the monolayer by apical extrusion. This reflects an intrinsic circuit where ATP released from apoptotic cells stimulates epithelial cells in the immediate vicinity to produce IL-8. As the epithelial barrier is compromised when apical extrusion fails, this juxta-apoptotic proinflammatory pathway may represent an early-response mechanism at sites of potential microbial ingress. Conversely, apical extrusion prevents inappropriate epithelial inflammation by physically eliminating apoptotic cells before they can activate this proinflammatory circuit.

cell biology↗

bioRxiv2021

Enhanced RhoA signaling stabilizes E-cadherin in migrating epithelial monolayers.

Epithelia migrate as physically coherent populations of cells. Earlier studies revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell-cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also reflects an increase in RhoA-mediated junctional contractility. We find that active RhoA levels were stimulated by p114 RhoGEF at the junctions between migrating MCF-7 monolayers, and this is accompanied by increased levels of actomyosin and mechanical tension. By applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin during epithelial migration. We suggest that stabilization of E-cadherin by RhoA serves to increase cell-cell adhesion against the mechanical stresses of migration.

cell biology↗

bioRxiv2020

A unified view of neighbour cell engagement during apoptotic cell extrusion.

Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation [1]. Several different mechanisms exist for apoptotic clearance, including efferocytosis [2, 3] and apical extrusion [4, 5]. We found that extrusion was the first-line response to apoptosis in cultured monolayers and in zebrafish epidermis. During extrusion, the apoptotic cell elicited active lamellipodial protrusions and assembly of a contractile extrusion ring in its neighbours. Depleting E-cadherin compromised both the contractile ring and extrusion, implying that a cadherin-dependent pathway allows apoptotic cells to engage their neighbours for extrusion. We identify RhoA as the cadherin-dependent signal in the neighbour cells and show that it is activated in response to contractile tension from the apoptotic cell. This mechanical stimulus is conveyed by a Myosin VI-dependent mechanotransduction pathway that is necessary both for extrusion and to preserve the epithelial barrier when apoptosis was stimulated. Earlier studies suggested that release of sphingosine-1-phosphate (S1P) from apoptotic cells might define where RhoA was activated. However, we found that although S1P is necessary for extrusion, its contribution does not require a localized source of S1P in the epithelium. We therefore propose a unified view of how RhoA is stimulated to engage neighbour cells for apoptotic extrusion. Here, tension-sensitive mechanotransduction is the proximate mechanism that activates RhoA specifically in the immediate neighbours of apoptotic cells, but this also must be primed by S1P in the tissue environment. Together, these elements provide a coincidence detection system that confers robustness on the extrusion response.

cell biology↗