ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Early embryonic polarity establishment and implications for lineage differentiation.

Polarity establishment is one of the key factors affecting early embryonic development. Polarity establishment begins with myosin phosphorylation in the 8-cell embryo, and phosphorylation activates actin leading to its initiation of contractility. Subsequently, actin undergoes reorganization to form an apical domain rich in microvilli on the non-contacting surface of each blastomere, and form the actomyosin ring that marks the maturation of the apical domain in conjunction with polar protein complexes and others. From the process of polarity establishment, it can be seen that the formation of the apical domain is influenced by actin-related proteins and polar protein complexes. Some zygote genome activation (ZGA) and lineage-specific genes also regulate polarity establishment. Polarity establishment underlies the first cell lineage differentiation during early embryonic development. It regulates lineage segregation and morphogenesis by affecting asymmetric cell division, asymmetric localization of lineage differentiation factors, and activity of the Hippo signaling pathway. In this review, we systematically summarize the mechanisms of early embryonic polarity establishment and its impact on lineage differentiation in mammals, and discuss the shortcomings of the currently available studies in terms of regulatory mechanisms and species, thereby providing clues and systematic perspectives for elucidating early embryonic polarity establishment.

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis.

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

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Objective: To investigate the synergistic regulation of the polarization of mesenchymal stem cells by integrin and N-cadherin-mediated mechanical adhesion and the underlying mechanobiological mechanisms. Methods: Bilayer polyethylene glyeol (PEG) hydrogels were formulated and modified with RGD and HAVDI peptides, respectively, to achieve mechanical adhesion to integrin and N-cadherin and to replicate the integrin-mediated mechanical interaction between cells and the extracellular matrix and the N-cadherin-mediated cell-cell mechanical interaction. The polar proteins, phosphatidylinositol 3-kinase (PI3K) and phosphorylated myosin light chain (pMLC), were characterized through immunofluorescence staining in individual cells with or without contact with HAVDI peptides under integrin-mediated adhesion, N-cadherin-mediated adhesion, and different intracellular forces. Their expression levels and polar distribution were analyzed using Image J. Results: Integrin-mediated adhesion induced significantly higher polar strengths of PI3K and pMLC in the contact group than in those in the no contact group, resulting in the concentration of the polar angle of PI3K to ß-catenin in the range of 135° to 180° and the concentration of the polar angle of pMLC to ß-catenin in the range of 0° to 45° in the contact group. Inhibition of integrin function led to inhibition of the polarity distribution of PI3K in the contact group, but did not change the polarity distribution of pMLC protein. The effect of N-cadherin on the polarity distributions of PI3K and pMLC was similar to that of integrin. However, inhibition of the mechanical adhesion of N-cadherin led to inhibition of the polarity intensity and polarity angle distribution of PI3K and pMLC proteins in the contact group. Furthermore, inhibition of the mechanical adhesion of N-cadherin caused weakened polarity intensity of integrin ß1, reducing the proportion of cells with polarity angles between integrin ß1 and ß-catenin concentrating in the range of 135° to 180°. Additionally, intracellular forces influenced the polar distribution of PI3K and pMLC proteins. Reducing intracellular forces weakened the polarity intensity of PI3K and pMLC proteins and their polarity distribution, while increasing intracellular forces enhanced the polarity intensity of PI3K and pMLC proteins and their polarity distribution. Conclusion: Integrin and N-cadherin co-regulate the polarity distribution of cell proteins and N-cadherin can play an important role in the polarity regulation of stem cells through local inhibition of integrin.

The functional role of L-fucose on dendritic cell function and polarization.

Despite significant advances in the development and refinement of immunotherapies administered to combat cancer over the past decades, a number of barriers continue to limit their efficacy. One significant clinical barrier is the inability to mount initial immune responses towards the tumor. As dendritic cells are central initiators of immune responses in the body, the elucidation of mechanisms that can be therapeutically leveraged to enhance their functions to drive anti-tumor immune responses is urgently needed. Here, we report that the dietary sugar L-fucose can be used to enhance the immunostimulatory activity of dendritic cells (DCs). L-fucose polarizes immature myeloid cells towards specific DC subsets, specifically cDC1 and moDC subsets. In vitro, L-fucose treatment enhances antigen uptake and processing of DCs. Furthermore, our data suggests that L-fucose-treated DCs increase stimulation of T cell populations. Consistent with our functional assays, single-cell RNA sequencing of intratumoral DCs from melanoma- and breast tumor-bearing mice confirmed transcriptional regulation and antigen processing as pathways that are significantly altered by dietary L-fucose. Together, this study provides the first evidence of the ability of L-fucose to bolster DC functionality and provides rational to further investigate how L-fucose can be used to leverage DC function in order to enhance current immunotherapy.

Exploring the role of CITED transcriptional regulators in the control of macrophage polarization.

Macrophages are tissue resident innate phagocytic cells that take on contrasting phenotypes, or polarization states, in response to the changing combination of microbial and cytokine signals at sites of infection. During the opening stages of an infection, macrophages adopt the proinflammatory, highly antimicrobial M1 state, later shifting to an anti-inflammatory, pro-tissue repair M2 state as the infection resolves. The changes in gene expression underlying these transitions are primarily governed by nuclear factor kappaB (NF-κB), Janus kinase (JAK)/signal transducer and activation of transcription (STAT), and hypoxia-inducible factor 1 (HIF1) transcription factors, the activity of which must be carefully controlled to ensure an effective yet spatially and temporally restricted inflammatory response. While much of this control is provided by pathway-specific feedback loops, recent work has shown that the transcriptional co-regulators of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxy-terminal domain (CITED) family serve as common controllers for these pathways. In this review, we describe how CITED proteins regulate polarization-associated gene expression changes by controlling the ability of transcription factors to form chromatin complexes with the histone acetyltransferase, CBP/p300. We will also cover how differences in the interactions between CITED1 and 2 with CBP/p300 drive their contrasting effects on pro-inflammatory gene expression.

Developmental progression continues during embryonic diapause in the roe deer.

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.

Inverted apicobasal polarity in health and disease.

Apicobasal epithelial polarity controls the functional properties of most organs. Thus, there has been extensive research on the molecular intricacies governing the establishment and maintenance of cell polarity. Whereas loss of apicobasal polarity is a well-documented phenomenon associated with multiple diseases, less is known regarding another type of apicobasal polarity alteration - the inversion of polarity. In this Review, we provide a unifying definition of inverted polarity and discuss multiple scenarios in mammalian systems and human health and disease in which apical and basolateral membrane domains are interchanged. This includes mammalian embryo implantation, monogenic diseases and dissemination of cancer cell clusters. For each example, the functional consequences of polarity inversion are assessed, revealing shared outcomes, including modifications in immune surveillance, altered drug sensitivity and changes in adhesions to neighboring cells. Finally, we highlight the molecular alterations associated with inverted apicobasal polarity and provide a molecular framework to connect these changes with the core cell polarity machinery and to explain roles of polarity inversion in health and disease. Based on the current state of the field, failure to respond to extracellular matrix (ECM) cues, increased cellular contractility and membrane trafficking defects are likely to account for most cases of inverted apicobasal polarity.

Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and the generation of cellular diversity. In the Caenorhabditis elegans zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, whereas in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together, these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.

The Drosophila neuroblast polarity cycle at a glance.

Drosophila neural stem cells, or neuroblasts, rapidly proliferate during embryonic and larval development to populate the central nervous system. Neuroblasts divide asymmetrically to create cellular diversity, with each division producing one sibling cell that retains the neuroblast fate and another that differentiates into glia or neurons. This asymmetric outcome is mediated by the transient polarization of numerous factors to the cell cortex during mitosis. The powerful genetics and outstanding imaging tractability of the neuroblast make it an excellent model system for studying the mechanisms of cell polarity. This Cell Science at a Glance article and the accompanying poster explore the phases of the neuroblast polarity cycle and the regulatory circuits that control them. We discuss the key features of the cycle - the targeted recruitment of proteins to specific regions of the plasma membrane and multiple phases of highly dynamic actomyosin-dependent cortical flows that pattern both protein distribution and membrane structure.

Distinct dynamics and proximity networks of hub proteins at the prey-invading cell pole in a predatory bacterium.

In bacteria, cell poles function as subcellular compartments where proteins localize during specific lifecycle stages, orchestrated by polar "hub" proteins. Whereas most described bacteria inherit an "old" pole from the mother cell and a "new" pole from cell division, generating cell asymmetry at birth, non-binary division poses challenges for establishing cell polarity, particularly for daughter cells inheriting only new poles. We investigated polarity dynamics in the obligate predatory bacterium Bdellovibrio bacteriovorus, proliferating through filamentous growth followed by non-binary division within prey bacteria. Monitoring the subcellular localization of two proteins known as polar hubs in other species, RomR and DivIVA, revealed RomR as an early polarity marker in B. bacteriovorus. RomR already marks the future anterior poles of the progeny during the predator's growth phase, during a precise period closely following the onset of divisome assembly and the end of chromosome segregation. In contrast to RomR's stable unipolar localization in the progeny, DivIVA exhibits a dynamic pole-to-pole localization. This behavior changes shortly before the division of the elongated predator cell, where DivIVA accumulates at all septa and both poles. In vivo protein interaction networks for DivIVA and RomR, mapped through endogenous miniTurbo-based proximity labeling, further underscore their distinct roles in cell polarization and reinforce the importance of the anterior "invasive" cell pole in prey-predator interactions. Our work also emphasizes the precise spatiotemporal order of cellular processes underlying B. bacteriovorus proliferation, offering insights into the subcellular organization of bacteria with filamentous growth and non-binary division.IMPORTANCEIn bacteria, cell poles are crucial areas where "hub" proteins orchestrate lifecycle events through interactions with multiple partners at specific times. While most bacteria exhibit one "old" and one "new" pole, inherited from the previous division event, setting polar identity poses challenges in bacteria with non-binary division. This study explores polar proteins in the predatory bacterium Bdellovibrio bacteriovorus, which undergoes filamentous growth followed by non-binary division inside another bacterium. Our research reveals distinct localization dynamics of the polar proteins RomR and DivIVA, highlighting RomR as an early "hub" marking polar identity in the filamentous mother cell. Using miniTurbo-based proximity labeling, we uncovered their unique protein networks. Overall, our work provides new insights into the cell polarity in non-binary dividing bacteria.

Perspectives on polarity - exploring biological asymmetry across scales.

In this Perspective, Journal of Cell Science invited researchers working on cell and tissue polarity to share their thoughts on unique, emerging or open questions relating to their field. The goal of this article is to feature 'voices' from scientists around the world and at various career stages, to bring attention to innovative and thought-provoking topics of interest to the cell biology community. These voices discuss intriguing questions that consider polarity across scales, evolution, development and disease. What can yeast and protists tell us about the evolution of cell and tissue polarity in animals? How are cell fate and development influenced by emerging dynamics in cell polarity? What can we learn from atypical and extreme polarity systems? How can we arrive at a more unified biophysical understanding of polarity? Taken together, these pieces demonstrate the broad relevance of the fascinating phenomenon of cell polarization to diverse fundamental biological questions.

Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression.

During asymmetric division of Drosophila larval neuroblasts, the fate determinant Prospero (Pros) and its adaptor Miranda (Mira) are segregated to the basal cortex through atypical protein kinase C (aPKC) phosphorylation of Mira and displacement from the apical cortex, but Mira localization after aPKC phosphorylation is not well understood. We identify Kin17, a DNA replication and repair protein, as a regulator of Mira localization during asymmetric cell division. Loss of Kin17 leads to aberrant localization of Mira and Pros to the centrosome, cytoplasm, and nucleus. We provide evidence to show that the mislocalization of Mira and Pros is likely due to reduced expression of Falafel (Flfl), a component of protein phosphatase 4 (PP4), and defects in dephosphorylation of serine-96 of Mira. Our work reveals that Mira is likely dephosphorylated by PP4 at the centrosome to ensure proper basal localization of Mira after aPKC phosphorylation and that Kin17 regulates PP4 activity by regulating Flfl expression.

Imaging and Analysis of Drosophila Neural Stem Cell Asymmetric Division.

Cell division is a conserved process among eukaryotes. It is designed to segregate chromosomes into future daughter cells and involves a complex rearrangement of the cytoskeleton, including microtubules and actin filaments. An additional level of complexity is present in asymmetric dividing stem cells because cytoskeleton elements are also regulated by polarity cues. The neural stem cell system of the fruit fly represents a simple model to dissect the mechanisms that control cytoskeleton reorganization during asymmetric division. In this chapter, we propose to describe protocols that allow accurate analysis of microtubule reorganization during cell division in this model.

A multiomics dataset for the study of RNA modifications in human macrophage differentiation and polarisation.

RNA modifications have emerged as central regulators of gene expression programs. Amongst RNA modifications are N6-methyladenosine (m6A) and RNA 5-hydroxymethylcytosine (5hmC). While m6A is established as a versatile regulator of RNA metabolism, the functions of RNA 5hmC are unclear. Despite some evidence linking RNA modifications to immunity, their implications in gene expression control in macrophage development and functions remain unclear. Here we present a multi-omics dataset capturing different layers of the gene expression programs driving macrophage differentiation and polarisation. We obtained mRNA-Seq, m6A-IP-Seq, 5hmC-IP-Seq, Polyribo-Seq and LC-MS/MS data from monocytes and resting-, pro- and anti-inflammatory-like macrophages. We present technical validation showing high quality and correlation between samples for all datasets, and evidence of biological consistency of modelled macrophages at the transcriptomic, epitranscriptomic, translational and proteomic levels. This multi-omics dataset provides a resource for the study of RNA m6A and 5hmC in the context of macrophage biology and spans the gene expression process from transcripts to proteins.

The Cyst Epithelium in Polycystic Kidney Disease Patients Displays Normal Apical-Basolateral Cell Polarity.

The main characteristic of polycystic kidney disease is the development of multiple fluid-filled renal cysts. The discovery of mislocalized sodium-potassium pump (Na,K-ATPase) in the apical membrane of cyst-lining epithelia alluded to reversal of polarity as a possible explanation for the fluid secretion. The topic of apical Na,K-ATPase in cysts remains controversial. We investigated the localization of the Na,K-ATPase and assessed the apical-basolateral polarization of cyst-lining epithelia by means of immunohistochemistry in kidney tissue from six polycystic kidney disease patients undergoing nephrectomy. The Na,K-ATPase α1 subunit was conventionally situated in the basolateral membrane of all immunoreactive cysts. Proteins of the Crumbs and partitioning defective (Par) complexes were localized to the apical membrane domain in cyst epithelial cells. The apical targeting protein Syntaxin-3 also immunolocalized to the apical domain of cyst-lining epithelial cells. Proteins of the basolateral Scribble complex immunolocalized to the basolateral domain of cysts. Thus, no deviations from the typical epithelial distribution of basic cell polarity proteins were observed in the cysts from the six patients. Furthermore, we confirmed that cysts can originate from virtually any tubular segment with preserved polarity. In conclusion, we find no evidence of a reversal in apical-basolateral polarity in cyst-lining epithelia in polycystic kidney disease.

Nuclear VANGL2 Inhibits Lactogenic Differentiation.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

CELSR1, a core planar cell polarity protein, features a weakly adhesive and flexible cadherin ectodomain.

Planar cell polarity (PCP), essential to multicellular developmental processes, arises when cells polarize and align across tissues. Central to PCP is CELSR1, an atypical cadherin featuring a long ectodomain with nine extracellular cadherin (EC) repeats, a membrane adjacent domain (MAD10), and several characteristic adhesion GPCR domains. Cell-based aggregation assays have demonstrated CELSR1's homophilic adhesive nature, but mechanistic details are missing. Here, we investigate the possible adhesive properties and structures of CELSR1 EC repeats. Our bead aggregation assays do not support strong adhesion by EC repeats alone. Consistently, EC1-4 only dimerizes at high concentration in solution. Crystal structures of human CELSR1 EC1-4 and EC4-7 reveal typical folds and a non-canonical linker between EC5 and EC6. Simulations and experiments using EC4-7 indicate flexibility at EC5-6, and solution experiments show EC7-MAD10-mediated dimerization. Our results suggest weak homophilic adhesion by CELSR1 cadherin repeats and provide mechanistic insights into the structural determinants of CELSR1 function.

Magnetically Stimulated Integrin Binding Alters Cell Polarity and Affects Epithelial-Mesenchymal Plasticity in Metastatic Cancer Cells.

Inorganic nanoparticles (NPs) have been widely recognized for their stability and biocompatibility, leading to their widespread use in biomedical applications. Our study introduces a novel approach that harnesses inorganic magnetic nanoparticles (MNPs) to stimulate apical-basal polarity and induce epithelial traits in cancer cells, targeting the hybrid epithelial/mesenchymal (E/M) state often linked to metastasis. We employed mesocrystalline iron oxide MNPs to apply an external magnetic field, disrupting normal cell polarity and simulating an artificial cellular environment. These led to noticeable changes in the cell shape and function, signaling a shift toward the hybrid E/M state. Our research suggests that apical-basal stimulation in cells through MNPs can effectively modulate key cellular markers associated with both epithelial and mesenchymal states without compromising the structural properties typical of mesenchymal cells. These insights advance our understanding of how cells respond to physical cues and pave the way for novel cancer treatment strategies. We anticipate that further research and validation will be instrumental in exploring the full potential of these findings in clinical applications, ensuring their safety and efficacy.

Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.

Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. Here, we generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. We show that Tmem208 is broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, we identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, our study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity.