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Abstract Primary cilia are sensory organelles present in many cell types, partaking in various signaling processes. Primary cilia of pancreatic beta cells play pivotal roles in paracrine signaling and their dysfunction is linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of beta cell primary cilia by electron and expansion microscopy. These cilia are spatially confined within deep ciliary pockets or narrow spaces between cells, lack motility components and display an unstructured axoneme organization. Furthermore, we observe a plethora of beta cell cilia-cilia and cilia-cell interactions with other islet and non-islet cells. Most remarkably, we have identified and characterized axo-ciliary synapses between beta cell cilia and the cholinergic islet innervation. These findings highlight the beta cell cilia’s role in islet connectivity, pointing at their function in integrating islet intrinsic and extrinsic signals and contribute to understanding their significance in health and diabetes.

The functionality of photoreceptors, rods, and cones is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during the development and in adulthood of wild-type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify the number, size, shape, and fluorescence intensity of POS, retinal cryosections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify the maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in “cone photoreceptor function loss 1” mice. TEM images were used to establish the ultrastructural quantification method QuaPOS-TEM for the alignment of POS membranes. Images were analyzed using a custom-written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild-type and two inherited retinal degeneration (“retinal degeneration 19” and “rhodopsin knock-out”) mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume, and membrane coherency during wild-type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All the code used for the presented analysis is open source, including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in-depth characterization of POS on retinal sections in developmental studies, for disease modeling, or after therapeutic interventions affecting photoreceptors.

Summary: Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

Summary: Zebrafish regenerate their fins which involves a component of cell plasticity. It is currently unclear how regenerate cells divide labor to allow for appropriate growth and patterning. Here, we studied lineage relationships of fluorescence-activated cell sorting-enriched epidermal, bone-forming (osteoblast), and (non-osteoblast) blastemal fin regenerate cells by single-cell RNA sequencing, lineage tracing, targeted osteoblast ablation, and electron microscopy. Most osteoblasts in the outgrowing regenerate derive from osterix+ osteoblasts, while mmp9+ cells reside at segment joints. Distal blastema cells contribute to distal osteoblast progenitors, suggesting compartmentalization of the regenerating appendage. Ablation of osterix+ osteoblasts impairs segment joint and bone matrix formation and decreases regenerate length which is partially compensated for by distal regenerate cells. Our study characterizes expression patterns and lineage relationships of rare fin regenerate cell populations, indicates inherent detection and compensation of impaired regeneration, suggests variable dependence on growth factor signaling, and demonstrates zonation of the elongating fin regenerate.

Objective: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. Methods: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) β cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. Results: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-β cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-β cell proliferation but had no impact on the expansion of SC-α cells. Conclusions: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.

Complex neuropathologies, like macular degeneration, are common but incompletely understood. Here the authors report a complex human retina pathology organoid model induced by HBEGF-TNF, causing PIEZO1-mediated photoreceptor degeneration by cell extrusion.

Spermatogenesis is a crucial biological process that enables the production of functional sperm, allowing for successful reproduction. Proper germ cell differentiation and maturation require tight regulation of hormonal signals, cellular signaling pathways, and cell biological processes. The acrosome is a lysosome-related organelle at the anterior of the sperm head that contains enzymes and receptors essential for egg-sperm recognition and fusion. Even though several factors crucial for acrosome biogenesis have been discovered, the precise molecular mechanism of pro-acrosomal vesicle formation and fusion is not yet known. In this study, we investigated the role of the insulin inhibitory receptor (inceptor) in acrosome formation. Inceptor is a single-pass transmembrane protein with similarities to mannose-6-phosphate receptors (M6PR). Inceptor knockout male mice are infertile due to malformations in the acrosome and defects in the nuclear shape of spermatozoa. We show that inceptor is expressed in early spermatids and mainly localizes to vesicles between the Golgi apparatus and acrosome. Here we show that inceptor is an essential factor in the intracellular transport of trans-Golgi network-derived vesicles which deliver acrosomal cargo in maturing spermatids. The absence of inceptor results in vesicle-fusion defects, acrosomal malformation, and male infertility. These findings support our hypothesis of inceptor as a universal lysosomal or lysosome-related organelle sorting receptor expressed in several secretory tissues.

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid–derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

The need for multifunctional materials for tissue engineering applications requires the development of multicomponent systems. Here, the authors report on the creation of a liquid metal-containing hydrogel with multiple covalent and noncovalent bonds to produce a tailorable, biocompatible biomaterial.

Elongation of the posterior body axis is distinct from that of the anterior trunk and head. Early drivers of posterior elongation are the neural plate/tube and notochord, later followed by the presomitic mesoderm (PSM), together with the neural tube and notochord. In axolotl, posterior neural plate-derived PSM is pushed posteriorly by convergence and extension of the neural plate. The PSM does not go through the blastopore but turns anteriorly to join the gastrulated paraxial mesoderm. To gain a deeper understanding of the process of axial elongation, a detailed characterization of PSM morphogenesis, which precedes somite formation, and of other tissues (such as the epidermis, lateral plate mesoderm and endoderm) is needed. We investigated these issues with specific tissue labelling techniques (DiI injections and GFP+ tissue grafting) in combination with optical tissue clearing and 3D reconstructions. We defined a spatiotemporal order of PSM morphogenesis that is characterized by changes in collective cell behaviour. The PSM forms a cohesive tissue strand and largely retains this cohesiveness even after epidermis removal. We show that during embryogenesis, the PSM, as well as the lateral plate and endoderm move anteriorly, while the net movement of the axis is posterior.

In T cells, processes such as migration and immunological synapse formation are accompanied by the dynamic reorganization of the actin cytoskeleton, which has been suggested to be mediated by regulators of RhoGTPases and by F-actin bundlers. SWAP-70 controls F-actin dynamics in various immune cells, but its role in T cell development and function has remained incompletely understood. CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 employ diverse mechanisms to suppress innate and adaptive immunity, which is critical for maintaining immune homeostasis and self-tolerance. Here, we propose Swap-70 as a novel member of the Foxp3-dependent canonical Treg cell signature. We show that Swap-70-/- mice have increased numbers of Foxp3+ Treg cells with an effector/memory-like phenotype that exhibit impaired suppressor function in vitro, but maintain overall immune homeostasis in vivo. Upon formation of an immunological synapse with antigen presenting cells in vitro, cytosolic SWAP-70 protein is selectively recruited to the interface in Treg cells. In this context, Swap-70-/- Treg cells fail to downregulate CD80/CD86 on osteoclast precursor cells by trans-endocytosis and to efficiently suppress osteoclastogenesis and osteoclast function. These data provide first evidence for a crucial role of SWAP-70 in Treg cell biology and further highlight the important non-immune function of Foxp3+ Treg cells in bone homeostasis mediated through direct SWAP-70-dependent mechanisms.

Summary: Maintenance of a healthy photoreceptor-retinal pigment epithelium (RPE) interface is essential for vision. At the center of this interface, apical membrane protrusions stemming from the RPE ensheath photoreceptor outer segments (POS), and are possibly involved in the recycling of POS through phagocytosis. The molecules that regulate POS ensheathment and its relationship to phagocytosis remain to be deciphered. By means of ultrastructural analysis, we revealed that Mer receptor tyrosine kinase (MERTK) ligands, GAS6 and PROS1, rather than αVβ5 integrin receptor ligands, triggered POS ensheathment by human embryonic stem cell (hESC)-derived RPE. Furthermore, we found that ensheathment is required for POS fragmentation before internalization. Consistently, POS ensheathment, fragmentation, and internalization were abolished in MERTK mutant RPE, and rescue of MERTK expression in retinitis pigmentosa (RP38) patient RPE counteracted these defects. Our results suggest that loss of ensheathment due to MERTK dysfunction might contribute to vision impairment in RP38 patients. : Using ultrastructural scanning electron microscopy (SEM)-based analysis, Almedawar et al. show that MERTK ligands, GAS6 and PROS1, upregulate photoreceptor outer segments (POS) ensheathment in human embryonic stem cell-derived retinal pigment epithelial cells. The authors also demonstrate that MERTK is essential for POS ensheathment and fragmentation before internalization, and suggest that ensheathment defects contribute to the pathology of retinitis pigmentosa type 38. Keywords: retinal pigment epithelium, photoreceptor outer segments, human embryonic stem cells, human pluripotent stem cells, phagocytosis, ensheathment, MERTK, GAS6, MFGE8, PROS1

Using retinal organoid systems, organ-like 3D tissues, relies implicitly on their robustness. However, essential key parameters, particularly retinal growth and longer-term culture, are still insufficiently defined. Here, we hypothesize that a previously optimized protocol for high yield of evenly-sized mouse retinal organoids with low variability facilitates assessment of such parameters. We demonstrate that these organoids reliably complete retinogenesis, and can be maintained at least up to 60 days in culture. During this time, the organoids continue to mature on a molecular and (ultra)structural level: They develop photoreceptor outer segments and synapses, transiently maintain its cell composition for about 5–10 days after completing retinogenesis, and subsequently develop pathologic changes – mainly of the inner but also outer retina and reactive gliosis. To test whether this organoid system provides experimental access to the retina during and upon completion of development, we defined and stimulated organoid growth by activating sonic hedgehog signaling, which in patients and mice in vivo with a congenital defect leads to enlarged eyes. Here, a sonic hedgehog signaling activator increased retinal epithelia length in the organoid system when applied during but not after completion of development. This experimentally supports organoid maturation, stability, and experimental reproducibility in this organoid system, and provides a potential enlarged retina pathology model, as well as a protocol for producing larger organoids. Together, our study advances the understanding of retinal growth, maturation, and maintenance, and further optimizes the organoid system for future utilization.

Changes in axonal myelination are an important hallmark of aging and a number of neurological diseases. Demyelinated axons are impaired in their function and degenerate over time. Oligodendrocytes, the cells responsible for myelination of axons, are sensitive to mechanical properties of their environment. Growing evidence indicates that mechanical properties of demyelinating lesions are different from the healthy state and thus have the potential to affect myelinating potential of oligodendrocytes. We performed a high-resolution spatial mapping of the mechanical heterogeneity of demyelinating lesions using atomic force microscope-enabled indentation. Our results indicate that the stiffness of specific regions of mouse brain tissue is influenced by age and degree of myelination. Here we specifically demonstrate that acquired acute but not genetic demyelination leads to decreased tissue stiffness, which could influence the remyelination potential of oligodendrocytes. We also demonstrate that specific brain regions have unique ranges of stiffness in white and grey matter. Our ex vivo findings may help the design of future in vitro models to mimic the mechanical environment of the brain in healthy and diseased states. The mechanical properties of demyelinating lesions reported here may facilitate novel approaches in treating demyelinating diseases such as multiple sclerosis. Statement of Significance: Mechanical characteristics of a cell's environment can have a profound influence on its biological properties. Neuronal and glial cells are sensitive to mechanical input during development, in disease and regeneration. Sustained tensile strain can promote differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes, which are responsible for the myelination of axons. Changing myelination is an important hallmark in human aging and disease, such as multiple sclerosis. Our hypothesis is that these diseases might be characterized by altered tissue stiffness and that this has an influence on remyelination potential. Here we investigate tissue stiffness profiles of healthy, aged and disease model mice. Manipulating the tissue stiffness might be another promising approach for new treatments.

The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (−37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Abstract Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Human brains are prone to neurodegeneration, given that endogenous neural stem/progenitor cells (NSPCs) fail to support neurogenesis. To investigate the molecular programs potentially mediating neurodegeneration-induced NSPC plasticity in regenerating organisms, we generated an Amyloid-β42 (Aβ42)-dependent neurotoxic model in adult zebrafish brain through cerebroventricular microinjection of cell-penetrating Aβ42 derivatives. Aβ42 deposits in neurons and causes phenotypes reminiscent of amyloid pathophysiology: apoptosis, microglial activation, synaptic degeneration, and learning deficits. Aβ42 also induces NSPC proliferation and enhanced neurogenesis. Interleukin-4 (IL4) is activated primarily in neurons and microglia/macrophages in response to Aβ42 and is sufficient to increase NSPC proliferation and neurogenesis via STAT6 phosphorylation through the IL4 receptor in NSPCs. Our results reveal a crosstalk between neurons and immune cells mediated by IL4/STAT6 signaling, which induces NSPC plasticity in zebrafish brains.

A central antiviral defence is immune recognition of cystolic DNA. Here the authors show that RPA and RAD51, in cooperation with TREX1, function to protect the cytosol from self-DNA.

Extracellular membrane vesicles (EVs) are emerging as new vehicles in intercellular communication, but how the biological information contained in EVs is shared between cells remains elusive. Several mechanisms have been described to explain their release from donor cells and the initial step of their uptake by recipient cells, which triggers a cellular response. Yet, the intracellular routes and subcellular fate of EV content upon internalization remain poorly characterized. This is particularly true for EV-associated proteins and nucleic acids that shuttle to the nucleus of host cells. In this review, we will describe and discuss the release of EVs from donor cells, their uptake by recipient cells, and the fate of their cargoes, focusing on a novel intracellular route wherein small GTPase Rab7+ late endosomes containing endocytosed EVs enter into nuclear envelope invaginations and deliver their cargo components to the nucleoplasm of recipient cells. A tripartite protein complex composed of (VAMP)-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3), and Rab7 is essential for the transfer of EV-derived components to the nuclear compartment by orchestrating the particular localization of late endosomes in the nucleoplasmic reticulum.