Biology subjects

Kurth, T.

Publications and source records attributed to Kurth, T..

At least 19 recordsLinked to original sources

bioRxiv2026

A tight balance of anabolic mTORC1 signaling and catabolic autophagic activity regulates zebrafish heart regeneration

Zebrafish can regenerate the heart by proliferation of cardiomyocytes. While the innate immune response and wound re-vascularization are pre-requisites for cardiomyocyte regeneration, little is known about signals linking early injury responses with the initiations of regenerative programs in cardiomyocytes. Here we show that mTOR (mechanistic target of rapamycin) signaling is rapidly activated in response to heart injury in many cell types of the heart including endothelial cells, cardiomyocytes and macrophages, but surprisingly not in neutrophils. We find that mTORC2 regulates macrophage recruitment to the wound, while mTORC1 is required for wound debris clearance by macrophages. In addition, mTOR signaling is required for wound re-vascularization. Interestingly, it also appears to directly regulate cardiomyocyte dedifferentiation and proliferation, making mTOR signaling a central hub for regenerative responses. Anabolic mTOR signaling acts as potent inhibitor of catabolic autophagy in many systems. Yet, we observed upregulation of autophagy within border zone cardiomyocytes where mTOR signaling is active. We show that mTOR signaling limits, but does not block autophagy, and that autophagic flux is regulated by both inhibitory mTOR signaling and stimulatory JNK and MEK pathways. Our results indicate that a fine-balanced anabolic and catabolic injury response is essential for zebrafish heart regeneration. Furthermore, they reveal interesting differences in the regulation of mTOR signaling and autophagy between the regenerative zebrafish heart and non-regenerative mammalian hearts.

developmental biology↗

bioRxiv2026

IDENTIFICATION OF HUMAN PHOTORECEPTORS SUITABLE FOR CELL REPLACEMENT STUDIES IN A PRECLINICAL ACHROMATOPSIA MODEL

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Muller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina. Significant statementCell transplantation represents a potential therapy for retinal dystrophies. However, conditions allowing stable functional integration and maturation of donor photoreceptors have not been defined. We systematically compared GMP-compliant iPSC-derived photoreceptors of different culture times after transplantation into a retinal degeneration mouse model. Photoreceptors of all ages showed structural integration and advanced maturation in close interaction with host Muller glia and bipolar cells, allowing restoration of light-driven responses. Thus human photoreceptors isolated from retinal organoids represent a robust source for clinical development of cell replacement in the diseased retina.

neuroscience↗

bioRxiv2026

Transplanted human photoreceptors differentially survive, incorporate, and mature in mildly and severely degenerated mouse retinae

Photoreceptor transplantation is considered a disease-agnostic therapeutic strategy for retinal degenerative diseases with highly heterogenous genetic, molecular, and cellular pathologies. While integration of human photoreceptors enriched from stem cell-derived retinal organoids was noted in previous preclinical studies, the potential influence of retinal degeneration severity on transplantation efficiency has not been systematically assessed. Here, we employed mice presenting mild or severe retinal degeneration as recipients for human induced pluripotent stem cell-derived photoreceptors. Donor cells formed multi-cellular clusters that structurally integrated from 3 weeks post-transplantation (wpt) in mildly degenerated retinas, closely interacting with host Muller glia, resulting in proper maturation characterized by inner/outer segment and synapse formation by 26 wpt. In contrast, in severely degenerated hosts, donor photoreceptors remained mainly singularized and scattered in the subretinal space, showing limited structural integration or signs of maturation. Differential maturation of donor cells in mild vs. severe hosts was confirmed by single-cell RNA-sequencing analysis. However, transplantation at the beginning of the degeneration process of the severe model allowed structural integration and maturation of donor photoreceptors, despite complete loss of endogenous photoreceptors over time. The study thus shows that survival, integration, and maturation of donor photoreceptors depend on the degenerative retinal microenvironment shaping significantly transplantation efficiency.

neuroscience↗

bioRxiv2025

NCBP2-AS2 is a mitochondrial microprotein, regulates energy metabolism and neurogenesis, and is downregulated in Alzheimer's disease

Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimers disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimers disease.

cell biology↗

bioRxiv2024

Extensive monolayer formation depends on a subpopulation of transplanted human iPSC-derived RPE cells

Loss of the retinal pigment epithelium (RPE) in the eye leads to photoreceptor dysfunction and death causing eventually vision loss. Cell replacement strategies using RPE cells derived in vitro from pluripotent stem cells (PSCs) are currently evaluated as a potential therapeutic strategy. Generation of polarized monolayers represents an essential prerequisite for proper RPE function, however, monolayer formation following transplantation of RPE cell suspensions has not been systematically assessed. Using the sodium iodate mouse model of acute RPE depletion, significant increase in monolayer formation capacity of passage (P) 1 vs. P2 human iPSC-derived RPE cells was observed three weeks after transplantation. Transplant-derived monolayers showed characteristic apicobasal polarity, RPE marker expression, phagocytosis function, and preservation of the host outer nuclear layer. The cell surface marker panel CD54+/PSA-NCAM- was identified to enrich for an RPE subpopulation with high potential for monolayer formation following transplantation. Results underline the importance of defining and isolating competent cell subpopulations for successful RPE transplantation.

neuroscience↗

bioRxiv2024

Somite-independent regeneration of the axolotl primary body axis

Primary body-axis development is a highly conserved process that proceeds through somitogenesis and subsequent subdivision into dermatome, myotome, and sclerotome. Defects in somitic-clock genes such as Hes7 lead to vertebral-segmentation defects in mice and fish. Here we show that in the axolotl, although Hes7 is necessary for proper embryonic vertebral segmentation, it is-- surprisingly--dispensable during tail regeneration. We investigated the mechanism of vertebral segmentation during regeneration which initially occurs through extension of a cartilage rod ventral to the spinal cord. We find that the regenerating cartilage rod undergoes a periodic wrinkling that provides a template for vertebral segmentation. Via direct mechanical measurements and biophysical perturbations, we show that a model of compression-induced buckling instability can predict vertebral segmentation. The cartilage rod and other somitic derivatives (muscle, cartilage, tendon, fibroblasts) arise from tendon-like, Lfng+ multi-potent mesenchymal progenitors, which display a gene regulatory state distinct from somitic progenitors. In summary, we uncover a mechanism of vertebral segmentation during axolotl tail regeneration that is distinct from the somite-based developmental mechanism.

developmental biology↗

bioRxiv2023

Structure, interaction, and nervous connectivity of beta cell primary cilia

Primary cilia are sensory organelles present in many cell types. Based on an array of microtubules termed axoneme they form a specialized membrane compartment partaking in various signaling processes. Primary cilia of pancreatic islet beta cells play a role in autocrine and paracrine signaling and are linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of complete mouse and human beta cell cilia, revealing a disorganized 9+0 axoneme structure. Within the islet, cilia are spatially confined within deep ciliary pockets or squeezed into narrow extracellular spaces between adjacent cells. Beta and alpha cell cilia physically interact with neighboring islet cells pushing and strongly bending their plasma membranes. Furthermore, beta cells can contain multiple cilia that can meet with other islet cell cilia in the extracellular space. Additionally, beta cell cilia establish connections with islet-projecting nerves. These findings highlight the pivotal role of beta cell primary cilia in islet cell connectivity, pointing at their potential functional role in integrating islet intrinsic and extrinsic signals. These novel insights contribute to understanding their significance in health and diabetes.

cell biology↗

bioRxiv2023

Fine-tuning of Fgf8 morphogen gradient by heparan sulfate proteoglycans in the extracellular matrix

Embryonic development is orchestrated by the action of morphogens, which spread out from a local source and activate, in a field of target cells, different cellular programs based on their concentration gradient. Fibroblast growth factor 8 (Fgf8) is a morphogen with important functions in embryonic organizing centers. It forms a gradient in the extracellular space by free diffusion, interaction with the extracellular matrix (ECM) and receptor-mediated endocytosis. However, morphogen gradient regulation by ECM is still poorly understood. Here we show that specific Heparan Sulfate Proteoglycans (HSPGs) bind Fgf8 directly in the ECM of living zebrafish embryos, thus affecting its diffusion and signaling. Using single-molecule Fluorescence Correlation Spectroscopy, we quantify this binding in vivo, and find two different modes of interaction. First, reducing or increasing the concentration of specific HSPGs in the extracellular space alters Fgf8 diffusion, and thus, its gradient shape. Second, ternary complex formation of Fgf8 ligand with Fgf-receptors and HSPGs at the cell surface requires HSPG attachment to the cell membrane. Together, our results show that graded Fgf8 morphogen distribution is achieved by constraining free Fgf8 diffusion through successive interactions with HSPGs at the cell surface and in ECM space. Statement of significanceFgf8 is a secreted morphogen signaling molecule that instructs neighboring arrays of undifferentiated cells about their position and cellular identity in tissue. Fgf8 and other morphogens are often distributed in a graded fashion, and can typically work at very low concentrations. To reproducibly generate information in developing tissue, mechanisms have evolved to carefully control distribution and concentration of Fgf8 morphogen. We show that freely diffusing Fgf8 morphogen moves through interstitial cell spaces on its way to target cells, and while doing so, interacts with ECM molecules in these spaces and at cell surfaces via low affinity, reversible binding. These interactions are important tuning mechanisms that contribute to forming the Fgf8 morphogen gradient and to cell surface receptor binding, and thus, to controlling cell type identity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/565243v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b6d073org.highwire.dtl.DTLVardef@2f64c1org.highwire.dtl.DTLVardef@17fbf19org.highwire.dtl.DTLVardef@1db334a_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractTo generate a gradient, the morphogen Fgf8 shuttles between fast free diffusion through extracellular space (i), slow diffusion or immobility (ii, iii) when bound to Heparan Sulfate Proteoglycans (HSPGs), in the extracellular matrix (ECM) and at cell surface receptors (iv), as revealed by single molecule studies in living zebrafish embryos.

developmental biology↗

bioRxiv2023

Rethinking animal attrition in preclinical research: expressing causal mechanisms of selection bias using directed acyclic graphs

Animal attrition in preclinical experiments can introduce bias in the estimation of causal treatment effects, as surviving animals may not be representative of the entire study population. This can compromise the internal validity of the study, despite randomization at the outset. Directed Acyclic Graphs (DAGs) are commonly used tools to transparently visualize assumptions about the causal structure underlying observed data. By illustrating relationships between relevant variables, DAGs enable the detection of even less intuitive biases, and can thereby inform strategies for their mitigation. In this study, we present an illustrative causal model for preclinical stroke research, in which animal attrition induces a specific type of selection bias (i.e., collider stratification bias) due to the interplay of animal welfare, initial disease severity and negative side effects of treatment. Even when the treatment had no causal effect, our simulations revealed substantial bias across different scenarios. We show how researchers can potentially mitigate this bias in the analysis phase, even when only data from surviving animals are available, if knowledge of the underlying causal process that gave rise to the data is available. Collider stratification bias should be a concern in preclinical animal model studies with severe side effects and high post-randomization attrition.

neuroscience↗

bioRxiv2023

Compartmentalization and synergy of osteoblasts drive bone formation in the regenerating fin

Zebrafish faithfully regenerate their fins after amputation which includes restoration of bone tissue and a component of cell plasticity. It is currently unclear how different cell populations of the regenerate divide labor to allow for efficient regenerate growth and proper patterning. Here, we studied lineage relationships of FACS-enriched epidermal, blastemal and bone forming fin regenerate cells by single cell (sc) RNA sequencing, lineage tracing, targeted osteoblast ablation and electron microscopy to show that the majority of osteoblasts in the outgrowing regenerate derive from osterix+ osteoblasts, while mmp9+ cells give rise to a limited cell number at the fin segment joints. A third population of distal blastema cells contributes to distal osteoblast progenitors, suggesting compartmentalization during appendage regeneration. Fin elongation and bone formation are carried out by distinct regenerate cell populations, and these variably depend on Fgf signaling. Ablation of osterix+ osteoblasts irreversibly impairs patterning of segment joints, and prevents bone matrix formation in the proximal regenerate. The resulting reduced regenerate length is partially compensated for by the distal regenerate which shows increased Wnt signaling activity. Surprisingly, ablation of joint cells does not abolish the formation of segment joints. Our study characterizes rare fin regenerate cell populations, indicates intricate osteoblast-blastema lineage relationships, inherent detection and compensation of impaired regeneration, and demonstrates zonation of the elongating regenerate. Furthermore, it sheds light on the variable dependence of bone formation on growth factor signaling.

developmental biology↗

bioRxiv2023

The effects of excess salt intake on the kidney metabolism in Sprague-Dawley rats

In the present study, novel methods were developed which allowed continuous (24/7) measurement of blood pressure (BP) and renal blood flow (RBF) in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. The study determined the effects of a high salt (HS) diet upon whole kidney O2 consumption and the metabolomic profiles of normal Sprague Dawley (SD) rats. A separate group of rats was studied to determine changes in the cortex (Cx) and outer medulla (OM) tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to a 4.0% NaCl diet. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. Increased glycolysis was evident with the elevation of mRNA expression encoding key glycolytic enzymes and release of pyruvate and lactate from the kidney in the renal venous blood. Glycolytic production of NADH is used in either the production of lactate or oxidized via the malate aspartate shuttle. Aerobic glycolysis (e.g., Warburg-effect) may account for the needed increase in cellular energy. The study provides evidence that kidney metabolism responds to a HS diet enabling enhanced energy production while protecting from oxidate stress and injury.

physiology↗

bioRxiv2022

Human photoreceptor cell transplants integrate into human retina organoids

Cell transplantation is a promising therapeutic approach to recover loss of neurons and vision in patient retinas. So far, human photoreceptor transplants restored some visual function in degenerating mouse retina. Whether retinal cell transplants also integrate into human retina, and how to optimize this for different pathologies are still unknown. Here, we sought to determine if human retina organoids generated from pluripotent stem cells might assist cell replacement therapy development in a human-to-human setting. Models for intra- and subretinal cell transplantation strategies were explored: Photoreceptor donor cells carrying a transgenic fluorescent reporter were enriched from acutely dissociated human retinal organoids. Donor cells were precisely transplanted by microinjection into the retina of host organoids, but high cell numbers might require multiple injections posing potential damage. Alternatively, donor cells were transplanted in large numbers by placing them in subretinal-like contact to the apical organoid surface. Using postmitotic retinal organoids (age >170-days) as a source for donor cells and as hosts, we show that six weeks after subretinal-like transplantation, large clusters of photoreceptors reproducibly incorporate into the host retina. Transplanted clusters frequently are located within or across the host photoreceptor layer, include cone and rod photoreceptors, and become infiltrated by cell processes of host Muller glia, indicative of structural integration. Histological and ultrastructural data of virally-labeled photoreceptor transplants show characteristic morphological and structural features of polarized photoreceptors: inner segments and ribbon synapses, and donor-host cell contacts develop contributing to the retinal outer limiting membrane. These results demonstrate that human retinal organoids provide a preclinical research system for cell replacement therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/500037v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@3a405dorg.highwire.dtl.DTLVardef@57c4deorg.highwire.dtl.DTLVardef@3de9c5org.highwire.dtl.DTLVardef@be9820_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

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 to clinical practice. Unlike the unstructured grafts of prior cell suspension transplantations into end-stage degeneration models, we describe extensive incorporation of 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, Muller cells extend throughout the graft, even forming a common outer limiting membrane. Donor-host interaction appears to promote polarisation as well as development of morphological features critical for light detection, namely formation of inner and well stacked outer segments oriented towards the RPE. Putative synapse formation and graft function is evident both at a structural and electrophysiological level. Overall, these results show that human photoreceptors interact readily with a partially degenerated retina. Moreover, incorporation into the host retina appears to be beneficial to graft maturation, polarisation and function. HighlightsO_LIGeneration of the first human iPSC cone reporter line C_LIO_LIHuman cones extensively incorporate into the retina of mice with cone degeneration C_LIO_LIDonor cone age and time in vivo are important factors for transplant incorporation C_LIO_LIIncorporation into the host retina correlates with graft polarisation C_LIO_LIImproved photoreceptor maturation after transplantation in vivo vs. in vitro C_LIO_LIRe-establishment of cone-mediated light-responses in the cone deficient mouse C_LI

neuroscience↗

bioRxiv2021

A single-cell atlas of de novo β-cell regeneration reveals the contribution of hybrid β/δ cells to diabetes recovery in zebrafish

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we use single-cell transcriptomics to map de novo {beta}-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing {delta}-cells, which we term {delta}1- and {delta}2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both {beta}- and {delta}1-cells. The transcriptomic analysis of {beta}-cell regeneration reveals that {beta}/{delta} hybrid cells constitute a prominent source of insulin-expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of {beta}-cell injury. Finally, interspecies comparison shows that plastic {delta}1-cells are partially related to PP-cells in the human pancreas. Our work provides an atlas of {beta}-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish

developmental biology↗

bioRxiv2021

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

{beta}-cells produce, store and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of {beta}-cell function is mostly derived from studies of ex vivo isolated islets and/or rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

cell biology↗

bioRxiv2020

AI-Driven Multiscale Simulations Illuminate Mechanisms of SARS-CoV-2 Spike Dynamics

We develop a generalizable AI-driven workflow that leverages heterogeneous HPC resources to explore the time-dependent dynamics of molecular systems. We use this workflow to investigate the mechanisms of infectivity of the SARS-CoV-2 spike protein, the main viral infection machinery. Our workflow enables more efficient investigation of spike dynamics in a variety of complex environments, including within a complete SARS-CoV-2 viral envelope simulation, which contains 305 million atoms and shows strong scaling on ORNL Summit using NAMD. We present several novel scientific discoveries, including the elucidation of the spikes full glycan shield, the role of spike glycans in modulating the infectivity of the virus, and the characterization of the flexible interactions between the spike and the human ACE2 receptor. We also demonstrate how AI can accelerate conformational sampling across different systems and pave the way for the future application of such methods to additional studies in SARS-CoV-2 and other molecular systems. ACM Reference FormatLorenzo Casalino1{dagger}, Abigail Dommer1{dagger}, Zied Gaieb1{dagger}, Emilia P. Barros1, Terra Sztain1, Surl-Hee Ahn1, Anda Trifan2,3, Alexander Brace2, Anthony Bogetti4, Heng Ma2, Hyungro Lee5, Matteo Turilli5, Syma Khalid6, Lillian Chong4, Carlos Simmerling7, David J. Hardy3, Julio D. C. Maia3, James C. Phillips3, Thorsten Kurth8, Abraham Stern8, Lei Huang9, John McCalpin9, Mahidhar Tatineni10, Tom Gibbs8, John E. Stone3, Shantenu Jha5, Arvind Ramanathan2*, Rommie E. Amaro1*. 2020. AI-Driven Multiscale Simulations Illuminate Mechanisms of SARS-CoV-2 Spike Dynamics. In Supercomputing 20: International Conference for High Performance Computing, Networking, Storage, and Analysis. ACM, New York, NY, USA, 14 pages. https://doi.org/finalDOI

biophysics↗

bioRxiv2020

Three-dimensional FIB-SEM reconstruction ofmicrotubule-organelle interaction in wholeprimary mouse beta cells

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is currently under debate. Here, we use FO_SCPLOWIBC_SCPLOW-SO_SCPLOWEMC_SCPLOW to image islet beta cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules and micro-tubules of seven beta cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that micro-tubules form non-radial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus supportive role in insulin secretion.

cell biology↗

bioRxiv2020

Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. The competence of secretory granules for exocytosis depends on spatio-temporal variables such as proximity to the plasma membrane and age, with newly-generated granules being preferentially released. Despite recent advances, we still lack a comprehensive view about the molecular composition of insulin granules and how this may change over the lifetime of the organelles. 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 obtained 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.

cell biology↗