Biology subjects

Kalaidzidis, Y.

Publications and source records attributed to Kalaidzidis, Y..

16 recordsLinked to original sources

bioRxiv2026

Reconstitution of lipid nanoparticle-mediated siRNA transfer across endosomal membrane mimics

Endosomal escape limits RNA delivery by lipid nanoparticles (LNPs). If escape requires membrane rupture, productive delivery would be intrinsically coupled to membrane damage and its potential toxicity. Here, we establish a cell-free system based on giant unilamellar vesicles (GUVs) with an early endosome-like lipid composition to dissect the mechanism of siRNA transfer and determine whether it can occur while membrane barrier function is preserved. Acidification promoted LNP-membrane association, lipid redistribution and siRNA translocation into the receiving lumen without detectable dissipation of transmembrane pH gradient. Complementary cryo-electron tomography revealed local interfacial lipid reorganisation without detectable membrane rupture. Fluorescence correlation spectroscopy revealed heterogeneous diffusion of translocated siRNA, consistent with free and lipid-associated species. Pre-formed RNA-lipid complexes exhibited pH-dependent dissociation and heterogeneous diffusion, supporting lipid association as a possible origin of slower luminal diffusion. Serum or apolipoprotein-E preincubation reduced siRNA translocation into the GUV lumen. Together, these findings support a model where siRNA-lipid association facilitates membrane crossing without sustained loss of barrier function. This platform provides a means to dissect LNP-mediated RNA delivery and develop formulations with improved efficacy while limiting membrane damage.

cell biology↗

bioRxiv2026

Chemical proteomics reveals post-translational remodeling of endosomal pathways during macrophage activation.

Macrophages are central regulators of innate immunity, yet the post-translational mechanisms that enable rapid inflammatory responses remain incompletely understood. Here, we integrated transcriptomics, unenriched proteomics, phosphoproteomics, metabolomics, and cysteine reactivity profiling to comprehensively characterize primary human macrophages following Toll-like receptor and Stimulator of Interferon Genes (STING) activation. This approach revealed substantial discordance between transcript- and protein-level changes, as well as extensive post-translational remodeling. Differentially reactive cysteines were enriched in proteins involved in small-GTPase signaling, vesicular trafficking, and the endo-lysosomal system. Reactivity changes in two subunits of the endosomal mRNA-transport FERRY complex nominated it as a candidate target of inflammation-associated post-translational regulation. Biochemical and imaging analyses established FERRY complex assembly and early endosomal localization in primary human macrophages and identified DISC1 as a previously unrecognized FERRY-associated protein. This work provides a comprehensive resource of biochemical and post-translational remodeling during macrophage activation and implicates endosome-associated mRNA transport in innate immune regulation.

immunology↗

bioRxiv2026

Villin-SNAP25 interaction regulates insulin secretion downstream of ICA512 signaling

Pancreatic islet {beta}-cells step-up insulin production and secretion in response to the elevation of blood glucose levels. ICA512/PTPRN is a transmembrane cargo of the insulin secretory granules (SGs). Upon SG exocytosis, its Ca2+/calpain mediated cleavage at the plasma membrane generates an ICA512-cleaved cytosolic fragment (ICA512-CCF) which as a decoy phosphatase prolongs phospho-STAT5/3 activities, thereby enhancing the transcription of mRNAs for SG cargoes, including insulin and its own, to replenish SG stores. In addition, ICA512 positively regulates the expression of F-actin modifier villin. Villin, in turn, modulates the size of actin cages surrounding cortical SGs, hence regulating SG mobility and exocytosis. Here we show that villin controls the number of SG docking sites for exocytosis by directly interacting at low glucose with the t-SNARE SNAP-25, thus restricting in this condition SG access to fusion sites and insulin release. Replacement of ICA512-CCF N-terminal ubiquitin-acceptor lysine 609 (K609) with valine (V) stabilized ICA512-CCF in insulinoma cells and homozygous ICA512K609V mice and enhanced the levels of phospho-STAT5/3 and villin. In ICA512K609V female mice these molecular traits correlated with reduced body weight, improved insulin sensitivity, reduced basal insulin secretion and onset time for glucose stimulated insulin secretion. Taken together, these data demonstrates that SG exocytosis induced ICA512 retrograde signaling acts in concert with the novel SNARE complex regulator villin to plastically adapt {beta}-cell actin cytoskeleton and access to SG docking sites for optimal control of insulin secretion in response to variations in extracellular glucose levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/699220v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@37d835org.highwire.dtl.DTLVardef@5389f1org.highwire.dtl.DTLVardef@855d98org.highwire.dtl.DTLVardef@1c3c639_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Model of ICA512 and STAT3/5-dependent regulation of -cell function. At basal conditions, villin limits secretory granule (SG) docking by interacting with SNAP-25, restricting insulin release (1). Increased metabolic load triggers hyperglycemia-induced ICA512 processing and growth factor-mediated STAT3/5 phosphorylation (2-4), enhancing ISG mRNAs, granule biogenesis, and villin expression (5,6). Villin modulates SNARE protein availability, controlling SG docking and secretion. Together, ICA512 retrograde signaling and villin-mediated SNARE regulation dynamically remodel the -cell actin cytoskeleton, adjusting SG docking site access to ensure appropriate insulin secretion in response to fluctuating glucose levels. C_FIG

cell biology↗

bioRxiv2025

RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

Metastasis is one of the primary causes of cancer-related death in Triple-negative breast cancer patients. During metastatic dissemination, the cancer cells infiltrate in to surrounding tissue employing specialized membrane-protrusion with proteolytic activity called invadopodia. Cues from growth factors via the cognate receptor tyrosine kinases promote invadopodia formation and cancer cell invasion. We report the role of HGF and its cognate receptor MET in TNBC invasion. The results from our study demonstrate that the TNBC cells when stimulated with HGF promotes invadopodia formation and facilitates delivery of MT1-MMP to the invadopodia in a MET dependent manner. We also showed that MET resides at invadopodia and its localization at invadopodia increases upon HGF treatment due to enhanced surface delivery of the RTK. By employing a degradation defective mutant, we demonstrated the critical role of MET recycling in driving breast cancer invasion. Moreover, we observed that the RCP-RAB14 axis mediates the surface delivery of MET through tubulovesicular carriers, with KIF16B promoting the formation of vesicular tubules on RAB14-positive endosomes. Collectively, the study provides mechanistic insights on the growth factor-mediated recycling of RTK and the associated metalloprotease in invadopodia-dependent matrix degradation and cancer cell invasion.

cancer biology↗

bioRxiv2025

GGA2-depletion in beta cells by Enteroviruses causes Golgi acidification, premature activation of cathepsins and alters the MHC class I immunopeptidome

Enteroviruses (EVs), such as Coxsackievirus B5 (CVB5), are linked to pancreatic beta cell autoimmunity in type 1 diabetes (T1D). CVB5 infection of beta cells blocks their cap-dependent translation but spares cap-independent translation of insulin secretory granule (SG) cargoes, including major T1D autoantigens. Despite this, insulin SG stores are depleted. We show that CVB5 protease 2A rapidly depletes Golgi-associated sorting factor GGA2 in beta cells due to its short half-life. Immunostaining of pancreas sections from recent-onset T1D donors confirmed reduced GGA2 in beta cells expressing EV capsid protein VP1. Both GGA2 depletion and CVB5 2A expression impair SG biogenesis at the TGN without affecting MHC class I trafficking. They also disrupt the sorting of vacuolar ATPase and cathepsins, leading to TGN acidification and premature lysosomal hydrolase activation, which typically generate peptides presented in HLA-II alleles. Notably, the unique immunopeptidome of EV-infected, GGA2-depleted ECN90 beta-like cells is mainly presented via HLA-B alleles with an average isoelectric point (pI) of [~]5, compared to a pI of [~]7 of the prevailing HLA-I A allele immunopeptidome of non-infected cells. We propose that delayed sorting at the acidified TGN promotes cathepsin-mediated processing of in-transit secretory proteins, including SG cargoes, thereby generating novel peptides that may replace HLA class I antigens due to the acidic environment. This novel cross-presentation mechanism could have implications for T1D pathogenesis and antigen presentation in general.

cell biology↗

bioRxiv2024

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi (BC). Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity common to epithelial cells. Whether and how cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Time-course RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feedback to hepatoblast-to-hepatocyte differentiation. Summary statementDifferentiation of liver progenitors to functional hepatocytes requires anisotropic elongation of their nascent apical surfaces into tubular bile canaliculi.

developmental biology↗

bioRxiv2023

Virtual tissue microstructure reconstruction by deep learning and fluorescence microscopy

Analyzing tissue microstructure is essential for understanding complex biological systems in different species. Tissue functions largely depend on their intrinsic tissue architecture. Therefore, studying the three-dimensional (3D) microstructure of tissues, such as the liver, is particularly fascinating due to its conserved essential roles in metabolic processes and detoxification. Here, we present TiMiGNet, a novel deep learning approach for virtual 3D tissue microstructure reconstruction using Generative Adversarial Networks (GANs) and fluorescence microscopy. TiMiGNet overcomes challenges such as poor antibody penetration and time-intensive procedures by generating accurate, high-resolution predictions of tissue components across large volumes without the need of paired images as input. We applied TiMiGNet to analyze tissue microstructure in mouse and human liver tissue. TiMiGNet shows high performance in predicting structures like bile canaliculi, sinusoids, and Kupffer cell shapes from actin meshwork images. Remarkably, using TiMiGNet we were able to computationally reconstruct tissue structures that cannot be directly imaged due experimental limitations in deep dense tissues, a significant advancement in deep tissue imaging. Our open-source virtual prediction tool facilitates accessible and efficient multi-species tissue microstructure analysis, accommodating researchers with varying expertise levels. TiMiGNets simplicity and independence from paired images make it a versatile asset. Overall, our method represents a powerful and efficient approach for studying tissue microstructure, with far-reaching applications in diverse biological contexts and species.

bioinformatics↗

bioRxiv2023

Hep3D: A 3D single-cell digital atlas of the liver to study spatio-temporal tissue architecture

Three dimensional (3D) geometrical models are not only a powerful tool for quantitatively characterizing complex tissues but also useful for probing structure-function relationships in a tissue. However, these models are generally incomplete due to experimental limitations in acquiring multiple (>4) fluorescent channels simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we performed deep-tissue immunostaining, multiphoton microscopy, deeplearning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatio-temporal singlecell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the structural role of KCs in the murine liver homeostasis. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs alters the number and morphology of the HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, under both physiological and pathological conditions.

cell biology↗

bioRxiv2021

The novel Rab5 effector FERRY links early endosomes with the translation machinery

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here we show that the novel Rab5 effector Five-subunit Endosomal Rab5 and RNA/ribosome intermediarY, FERRY complex recruits mRNAs and ribosomes to early endosomes, through direct mRNA interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA and protein levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

cell biology↗

bioRxiv2021

Bulkhead-like apical membrane structures between hepatocytes are required for anisotropic lumen expansion and liver tissue morphogenesis

Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces1-3. The mechanisms governing anisotropic lumen expansion remain elusive4-6. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC)7,8. Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis.

cell biology↗

bioRxiv2020

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those employed in mRNA-based vaccines, in primary human adipocytes, fibroblasts and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery and different LNP vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By super-resolution microscopy we could resolve single LNP-mRNA within sub-endosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity.

bioengineering↗

bioRxiv2019

Correlative SMLM and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which however cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional sub-domains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-colour SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.\n\nSynopsisSuborganelle compartmentalization cannot be resolved by diffraction limited light microscopy and not interpreted without knowledge of the underlying ultrastructure. This work shows a novel superCLEM workflow that combines multi-colour single-molecule localization-microscopy with electron tomography to map several functional domains on early endosomes. superCLEM reveals that the small GTPase Rab5 is organized in nano-domains largely devoid from cargo molecules Transferrin and EGF and opens new possibilities to perform structure-function analysis of organelles at the nanoscale.

cell biology↗

bioRxiv2019

Metabolic control of YAP via the acto-myosin system during liver regeneration

The mechanisms of organ size control remain poorly understood. A key question is how cells collectively sense the overall status of a tissue. We addressed this problem focusing on mouse liver regeneration, which is controlled by Hippo signalling. Using digital tissue reconstruction and quantitative image analysis, we found that the apical surface of hepatocytes forming the bile canalicular network expands concomitant with an increase of F-actin and phospho-Myosin, to compensate an overload of bile acids. Interestingly, these changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to the apical F-actin-rich region and translocates to the nucleus in dependence of the acto-myosin system. This mechanism tolerates moderate bile acid fluctuations under tissue homeostasis, but activates YAP in response to sustained bile acid overload. Using an integrated biophysical-biochemical model of bile pressure and Hippo signalling, we explained this behaviour by the existence of a mechano-sensory mechanism that activates YAP in a switch-like manner. We propose that the apical surface of hepatocytes acts as a self-regulatory mechano-sensory system that responds to critical levels of bile acids as readout of tissue status.

systems biology↗

bioRxiv2019

3D spatially-resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression

Early disease diagnosis is key for the effective treatment of diseases. It relies on the identification of biomarkers and morphological inspection of organs and tissues. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D structural changes that are consequence of functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially-resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of new morphometric cellular parameters correlated with disease progression. Moreover, we found profound topological defects in the 3D bile canaliculi (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and zonated cholestasis, consistent with elevated cholestatic biomarkers in patients sera. Our spatially-resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multi-parametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology.

pathology↗

bioRxiv2018

Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Long-distance axonal trafficking plays a critical role in neuronal function, and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

cell biology↗

bioRxiv2018

Liquid-crystal organization of liver tissue

Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells as generally assumed, since silencing Integrin-{beta}1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.

biophysics↗