Defective mitochondria remodelling in B cells leads to an aged immune response.

The B cell response in the germinal centre (GC) reaction requires a unique bioenergetic supply. Although mitochondria are remodelled upon antigen-mediated B cell receptor stimulation, mitochondrial function in B cells is still poorly understood. To gain a better understanding of the role of mitochondria in B cell function, here we generate mice with B cell-specific deficiency in Tfam, a transcription factor necessary for mitochondrial biogenesis. Tfam conditional knock-out (KO) mice display a blockage of the GC reaction and a bias of B cell differentiation towards memory B cells and aged-related B cells, hallmarks of an aged immune response. Unexpectedly, blocked GC reaction in Tfam KO mice is not caused by defects in the bioenergetic supply but is associated with a defect in the remodelling of the lysosomal compartment in B cells. Our results may thus describe a mitochondrial function for lysosome regulation and the downstream antigen presentation in B cells during the GC reaction, the dysruption of which is manifested as an aged immune response.

CartoCell, a high-content pipeline for 3D image analysis, unveils cell morphology patterns in epithelia.

Decades of research have not yet fully explained the mechanisms of epithelial self-organization and 3D packing. Single-cell analysis of large 3D epithelial libraries is crucial for understanding the assembly and function of whole tissues. Combining 3D epithelial imaging with advanced deep-learning segmentation methods is essential for enabling this high-content analysis. We introduce CartoCell, a deep-learning-based pipeline that uses small datasets to generate accurate labels for hundreds of whole 3D epithelial cysts. Our method detects the realistic morphology of epithelial cells and their contacts in the 3D structure of the tissue. CartoCell enables the quantification of geometric and packing features at the cellular level. Our single-cell cartography approach then maps the distribution of these features on 2D plots and 3D surface maps, revealing cell morphology patterns in epithelial cysts. Additionally, we show that CartoCell can be adapted to other types of epithelial tissues.

Cadherin-mediated adhesion takes control.

The formation of a centralised apical membrane initiation site (AMIS) is a key event in epithelial cell polarisation. A recent study by Liang et al demonstrates that AMIS localisation relies on cadherin-mediated cell adhesion.

Tip-end fusion of a rod-shaped secretory organelle.

Weibel-Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB-plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.

Deciphering the interplay between autophagy and polarity in epithelial tubulogenesis.

The Metazoan complexity arises from a primary building block, the epithelium, which comprises a layer of polarized cells that divide the organism into compartments. Most of these body compartments are organs formed by epithelial tubes that enclose an internal hollow space or lumen. Over the last decades, multiple studies have unmasked the paramount events required to form this lumen de novo. In epithelial cells, these events mainly involve recognizing external clues, establishing and maintaining apicobasal polarity, endo-lysosomal trafficking, and expanding the created lumen. Although canonical autophagy has been classically considered a catabolic process needed for cell survival, multiple studies have also emphasized its crucial role in epithelial polarity, morphogenesis and cellular homeostasis. Furthermore, non-canonical autophagy pathways have been recently discovered as atypical secretory routes. Both canonical and non-canonical pathways play essential roles in epithelial polarity and lumen formation. This review addresses how the molecular machinery for epithelial polarity and autophagy interplay in different processes and how autophagy functions influence lumenogenesis, emphasizing its role in the lumen formation key events.

Actomyosin fibers DApPLE epithelial apical junctions.

Epithelial cell morphology is essential for cellular homeostasis, but the mechanisms by which cell shape is established remain unclear. In this study, Marivin et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202111002) identify DAPLE as a linker between polarity complexes and the actomyosin network at apical junctions. By recruiting CD2P and activating Gαßγ-mediated RhoA signaling, DAPLE ensures proper cell shape and function.

Intercalate or invaginate: PI(3,4,5)P3 governs a membrane constriction switch in cell shaping.

Although contractile processes, from tissue invagination to cell intercalation, utilize diverse ratcheting mechanisms, little is known about how ratcheting becomes engaged at specific cell surfaces. In this issue of Developmental Cell, Maio et al. demonstrate that PI(3,4,5)P3 is a paramount regulator of the Sbf/RabGEF-Rab35 ratchet mechanism.

Methods to Generate Tube Micropatterns for Epithelial Morphogenetic Analyses and Tissue Engineering.

Cells live in a highly curved and folded 3D microenvironment within the human body. Since epithelial cells in internal organs usually adopt a tubular shape, there is a need to engineer simple in vitro devices to promote this cellular configuration. The aim of these devices would be to investigate epithelial morphogenesis and cell behavior-leading to the development of more sophisticated platforms for tissue engineering and regenerative medicine. In this chapter, we first explain the need for such epithelial tubular micropatterns based on anatomical considerations and then survey methods that can be used to study different aspects of epithelial tubulogenesis. The methods examined can broadly be divided into two classes: conventional 2D microfabrication for the formation of simple epithelial tubes in substrates of different stiffness; and 3D approaches to enable the self-assembly of organoid-derived epithelial tubes in a tubular configuration. These methods demonstrate that modeling tubulogenesis in vitro with high resolution, accuracy, and reproducibility is possible.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis.

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

The vertebrate epithelial apical junctional complex: Dynamic interplay between Rho GTPase activity and cell polarization processes.

Epithelial tissues are made of highly specialized cells present in many organs and represent the first barrier of protection from the external environment. Essential for this critical role in protection is their capacity to polarize in the apicobasal axis. The integrity of the epithelium and its properties as a protective barrier is mostly regulated by dynamic intercellular junctions composed of multiprotein complexes. The functionality and dynamics of these junctions are tightly controlled by several signaling processes, including Rho GTPases. Here, we review the most recent data in the contribution of Rho GTPases and their functional regulators during the morphogenesis of epithelial tissues and to maintain the homeostasis in adults.

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity.

Tubulogenesis in epithelial organs often initiates with the acquisition of apicobasal polarity, giving rise to the formation of small lumens that expand and fuse to generate a single opened cavity. In this study, we present a micropattern-based device engineered to generate epithelial tubes through a process that recapitulates in vivo tubule morphogenesis. Interestingly, tubulogenesis in this device is dependent on microenvironmental cues such as cell confinement, extracellular matrix composition, and substrate stiffness, and our set-up specifically allows the control of these extracellular conditions. Additionally, proximal tubule cell lines growing on micropatterns express higher levels of drug transporters and are more sensitive to nephrotoxicity. These tubes display specific morphological defects that can be linked to nephrotoxicity, which would be helpful to predict potential toxicity when developing new compounds. This device, with the ability to recapitulate tube formation in vitro, has emerged as a powerful tool to study the molecular mechanisms involved in organogenesis and, by being more physiologically relevant than existing cellular models, becomes an innovative platform to conduct drug discovery assays.

Sfrp3 modulates stromal-epithelial crosstalk during mammary gland development by regulating Wnt levels.

Mammary stroma is essential for epithelial morphogenesis and development. Indeed, postnatal mammary gland (MG) development is controlled locally by the repetitive and bi-directional cross-talk between the epithelial and the stromal compartment. However, the signalling pathways involved in stromal-epithelial communication are not entirely understood. Here, we identify Sfrp3 as a mediator of the stromal-epithelial communication that is required for normal mouse MG development. Using Drosophila wing imaginal disc, we demonstrate that Sfrp3 functions as an extracellular transporter of Wnts that facilitates their diffusion, and thus, their levels in the boundaries of different compartments. Indeed, loss of Sfrp3 in mice leads to an increase of ductal invasion and branching mirroring an early pregnancy state. Finally, we observe that loss of Sfrp3 predisposes for invasive breast cancer. Altogether, our study shows that Sfrp3 controls MG morphogenesis by modulating the stromal-epithelial cross-talk during pubertal development.

Mechanosensitive adhesion complexes in epithelial architecture and cancer onset.

Mechanical signals from the extracellular space are paramount to coordinate tissue morphogenesis and homeostasis. Although there is a wide variety of cellular mechanisms involved in transducing extracellular forces, recent literature emphasizes the central role of two main adhesion complexes in epithelial mechanosensitive processes: focal adhesions and adherens junctions. These biomechanical sensors can decode physical signals such as matrix stiffness or intercellular tension into a wide range of coordinated cellular responses, which can impact cell differentiation, migration, and proliferation. Communication between cells and their microenvironment plays a pivotal role both in physiological and pathological conditions. Here we summarize the most recent findings on the biology of these mechanotransduction pathways in epithelial cells, highlighting the extensive amount of biological processes coordinated by cell-matrix and cell-cell adhesion complexes.

Signaling Networks in Epithelial Tube Formation.

Epithelial tubes are crucial to the function of organ systems including the excretory, gastrointestinal, cardiovascular, and pulmonary. Studies in the last two decades using in vitro organotypic systems and a variety of animal models have substantiated a large number of the morphogenetic mechanisms required to form epithelial tubes in development and regeneration. Many of these mechanisms modulate the differentiation and proliferation events necessary for generating the cell movements and changes in cell shape to delineate the wide variety of epithelial tube sizes, lengths, and conformations. For instance, when coupled with oriented cell division, proliferation itself plays a role in changes in tube shape and their directed expansion. Most of these processes are regulated in response to signaling inputs from adjacent cells or soluble factors from the environment. Despite the great deal of recent investigation in this direction, the knowledge we have about the signaling pathways associated with all epithelial tubulogenesis in development and regeneration is still very limited.

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells.

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation.

Endocytic turnover of Rab8 controls cell polarization.

Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.

PRL-3 disrupts epithelial architecture by altering the post-mitotic midbody position.

Disruption of epithelial architecture is a fundamental event during epithelial tumorigenesis. We show that the expression of the cancer-promoting phosphatase PRL-3 (PTP4A3), which is overexpressed in several epithelial cancers, in polarized epithelial MDCK and Caco2 cells leads to invasion and the formation of multiple ectopic, fully polarized lumens in cysts. Both processes disrupt epithelial architecture and are hallmarks of cancer. The pathological relevance of these findings is supported by the knockdown of endogenous PRL-3 in MCF-7 breast cancer cells grown in three-dimensional branched structures, showing the rescue from multiple-lumen- to single-lumen-containing branch ends. Mechanistically, it has been previously shown that ectopic lumens can arise from midbodies that have been mislocalized through the loss of mitotic spindle orientation or through the loss of asymmetric abscission. Here, we show that PRL-3 triggers ectopic lumen formation through midbody mispositioning without altering the spindle orientation or asymmetric abscission, instead, PRL-3 accelerates cytokinesis, suggesting that this process is an alternative new mechanism for ectopic lumen formation in MDCK cysts. The disruption of epithelial architecture by PRL-3 revealed here is a newly recognized mechanism for PRL-3-promoted cancer progression.

Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair.

Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.