Biology subjects

Klumperman, J.

Publications and source records attributed to Klumperman, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Calsyntenin-1 and calsyntenin-3 coordinate TGN exit of axonal cargoes

Neurons rely on precise biosynthetic protein transport from the soma to the axon and dendrites. Brain-enriched calsyntenins (CSTN1-3) are unique transmembrane adaptors that link cargo to kinesin-1 for transport. Yet, the neuronal distribution and interdependence of the three CSTN paralogs remain unclear. Here, we dissected their subcellular localization and contribution to biosynthetic protein transport. CSTN paralogs were predominantly localized to the TGN and axonal vesicles, with CSTN1 and CSTN3 showing higher expression levels than CSTN2. Depletion of either CSTN1 or CSTN3 affected axonal abundance of the other, suggesting that they function within the same pathway. Consistently, knockdown of CSTN1 or CSTN3, but not CSTN2, impaired the TGN exit and transport of multiple biosynthetic cargoes to the axon. Interestingly, most CSTN-positive vesicles exiting the TGN and in the axon were marked by the biosynthetic trafficking regulator RAB6A. Furthermore, we identified opposing, paralog-specific roles of CSTN1 and CSTN3 in regulating RAB6A levels at the TGN, thereby contributing to Golgi organization and axonal trafficking. Loss of CSTN1 reduced RAB6A and induced Golgi compaction, whereas loss of CSTN3 increased RAB6A and promoted Golgi dispersal. Together, we reveal that CSTN1 and CSTN3 have distinct, yet intersecting roles in the regulation of biosynthetic axonal transport.

Cell Biology↗

bioRxiv2026

VPS41 loss triggers iron overload, oxidative stress, and mitochondrial fragmentation linked to ferroptosis

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

cell biology↗

bioRxiv2026

HOPS is required for non-canonical mTORC1 signaling by recruiting Rags and FLCN to lysosomal membranes

The mechanistic target of Rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to nutrient availability. mTORC1 recruitment to lysosomes by the Rag dimer complex (RagA/B and RagC/D) is a crucial step in mTORC1 signaling. Substrates of the MiT/TFE transcription factor family, like TFEB and TFE3, directly interact with the Rag complex when activated by folliculin (FLCN). This selective recruitment and subsequent phosphorylation of MiT/TFE substrates leads to a non-canonical mTORC1 signaling pathway regulating lysosomal biogenesis and autophagy. Recently, we showed that compound heterozygous mutations in VPS41 specifically impair non-canonical mTORC1 signaling. VPS41, as part of the HOPS complex, is required for fusion of lysosomes with endosomes and autophagosomes. Here we addressed the mechanism by which VPS41/HOPS complex regulates mTORC1 activity. We show that multiple HOPS subunits interact with the Rag dimers as well as with FLCN. Depletion of HOPS subunits results in reduced lysosomal localization of both Rags and FLCN, and the nuclear translocation of TFE3/TFEB. The VPS41-Rag interactions required the RING domain of VPS41, but were independent of RHEB, Rag activity or presence of other HOPS components. We conclude that HOPS is necessary for the recruitment of crucial components of the non-canonical mTORC1 signaling pathway onto lysosomal membranes. These data further our molecular understanding of disease-causing VPS41/HOPS mutations and indicate a crucial role for HOPS in connecting lysosomal trafficking to lysosomal signaling.

cell biology↗

bioRxiv2026

CD4+ T cell activation is dependent on a novel form of ULK1/2-independent autophagy

Autophagy is essential for CD4+ T cell activation and immune regulation. However, during activation both autophagy and anabolic signaling must be simultaneously sustained, challenging established models of pathway antagonism. Here, we show that T cell receptor signaling and co-stimulation induce a non-canonical form of autophagy required for proliferation and cytokine production. Pharmacological and genetic analyses reveal that this pathway is activated concurrently with mTORC1, and is dependent on PIK3C3, but occurs independently of the canonical regulators ULK1/2, AMPK, ATG13, and Beclin 1. Furthermore, immuno-electron microscopy demonstrates that activation generates smaller autophagic structures that associate with multivesicular bodies and exhibit a unique morphology. These findings uncover a fundamental rewiring of autophagy control in CD4+ T cells and identify a novel form of mechanistically and morphologically distinct non-canonical autophagy.

cell biology↗

bioRxiv2025

HOPS disruption impairs APP trafficking and processing, promoting exosomal secretion of APP-CTFs

Amyloid precursor protein (APP) is a key player in various neuronal functions but also the source for toxic A{beta} that accumulates in the brain of Alzheimer patients. APP trafficking and processing depend on the endo-lysosomal system, but the molecular mechanisms that coordinate these processes remain unclear. Here, we studied the HOPS complex, a central regulator of endo-lysosomal maturation. We show that HOPS disruption impairs retromer-mediated recycling of APP to the TGN, resulting in the accumulation of APP in late endosomes. In neurons, this accumulation is spatially restricted to somatodendritic endosomes. These APP-containing endosomes are catalytically inactive and lack the {gamma}-secretase subunit PSEN2. However, they do contain BACE1, which leads to the build-up of toxic APP C-terminal fragments (APP-CTFs) upon HOPS disruption. Notably, loss of HOPS enhances secretion of APP-CTFs by exosomes, suggesting a potential mechanism for disease propagation. Together, our findings establish a mechanistic link between HOPS dysfunction and aberrant APP processing, with implications for neurodegeneration. HighlightsO_LIHOPS KO impairs retromer-mediated APP recycling to the TGN C_LIO_LIHOPS disruption redistributes APP to somatodendritic stationary late endosomes C_LIO_LIHOPS depletion increases APP and BACE1 convergence, causing APP-CTF accumulation C_LIO_LIThese APP-CTFs accumulate in catalytically inactive endosomes that lack PSEN2 C_LIO_LIAPP-CTFs are secreted via exosomes, potentially promoting disease propagation C_LI

cell biology↗

bioRxiv2025

β-Catenin condensation facilitates clustering of the cadherin/catenin complex and formation of nascent cell-cell junctions

Cadherin-based junctions establish dynamically regulated adhesion between cells to coordinate tissue integrity and morphogenetic movements. Adhesion strength can be modulated by the organization of individual cadherin complexes into lateral clusters. Here, we identify a novel clustering mechanism of the cadherin complex established by its core component {beta}-catenin. We show that the disordered termini of {beta}-catenin drive the formation of condensates that incorporate other components of the cadherin complex in vitro. Using {beta}-catenin mutants with hampered condensation, we demonstrate that {beta}-catenin condensation nucleates the formation of submicron cadherin/catenin clusters that further develop into stable sites of adhesion. Furthermore, we show that {beta}-catenin-dependent clustering ensures the efficient formation of de novo cell-cell adhesions. Our data thus indicate a role for {beta}-catenin condensates in the supramolecular organization of the cadherin complex, and reveal that the function of {beta}-catenin in the cadherin complex extends beyond connecting cadherin to -catenin and the actin cytoskeleton.

cell biology↗

bioRxiv2025

Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

Legionella pneumophila is an intracellular pathogen that causes Legionnaires disease. The bacteria releases effector proteins some of which remodel the host autophagic-lysosomal pathways. One effector is RavZ, which delipidates ATG8 proteins, making Legionella-infected cells deficient in autophagy. Here we show that SidE effectors mediate the phosphoribosyl ubiquitination (PR-Ub) of autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17+ membranes from the endoplasmic reticulum to Legionella-containing phagosomes to form replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we have discovered a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole which has autophagy markers but do not fuse with lysosomes. Mass spectrometric identification of PR-Ub sites and mutational studies show that PR-Ub modification of STX17 alters its interaction with ATG14L, which drives the recruitment of ER membranes to the bacterial vacuole in a PI3K dependent manner. On the other hand, PR-Ub of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) by steric hindrance, thereby preventing the fusion of bacterial vacuoles with lysosomes.

cell biology↗

bioRxiv2025

Identification of a RAB32-LRMDA-Commander membrane trafficking complex reveals the molecular mechanism of human oculocutaneous albinism type 7

Commander is an endosome associated sixteen protein assembly that associates with the sorting nexin 17 (SNX17) cargo adaptor to regulate cell surface recycling of internalised integral membrane proteins including integrins and lipoprotein receptors. Mutations in Commander are causative for Ritscher-Schinzel syndrome (RSS), a multiorgan developmental disease associated with a core triad of cerebellar-cardiac-craniofacial malformation. Here, using unbiased proteomics and computational modelling, we identify leucine rich melanocyte differentiation associated (LRMDA) as a novel Commander binding protein. Using recombinant protein reconstitution, we show that LRMDA simultaneously associates with Commander and active RAB32, and, by revealing that LRMDA and SNX17 share a common mechanism of Commander association, establish the mutually exclusive nature of RAB32-LRMDA-Commander and SNX17-Commander assemblies. From functional analysis in human melanocytes, we establish an essential role for RAB32-LRMDA-Commander in melanosome biogenesis and pigmentation and reveal a distinct functional role for SNX17-Commander in this organelle biogenesis pathway. We reveal how LRMDA mutations, causative for oculocutaneous albinism type 7 (OCA7), a hypopigmentation disorder accompanied by poor visual acuity, uncouple RAB32 and Commander binding thereby establishing the mechanistic basis of this disease. Our discovery and characterisation of this alternative Commander assembly establishes an unrecognised plasticity of Commander function within a highly complex organelle biogenesis pathway. This extends Commander function beyond the confines of SNX17-mediated cell surface recycling into RAB32-family mediated biogenesis of lysosome-related organelles and, potentially, other RAB32 regulated pathways including host-pathogen defence mechanisms. Our work also extends the breath of Commander pathway dysfunction for human disease.

cell biology↗

bioRxiv2023

A nonenzymatic dependency on inositol-requiring enzyme 1 controls cancer cell cycle progression and tumor growth

Endoplasmic-reticulum resident inositol-requiring enzyme 1 (IRE1) supports protein homeostasis via a cytoplasmic kinase-RNase module. Known cancer dependency on IRE1 entails its enzymatic activation of the transcription factor XBP1s and of RNA decay. We discovered that some cancer cells require IRE1 but not its enzymatic activity. IRE1 knockdown, but not enzymatic inhibition or XBP1 disruption, increased DNA damage and chromosome instability while engaging the TP53 pathway and cyclin-dependent kinase inhibitors and attenuating cell cycle progression. IRE1 depletion downregulated factors involved in chromosome replication and segregation and in chromatin remodeling. Immunoelectron microscopy indicated that endogenous IRE1 can localize to the nuclear envelope. Thus, cancer cells can require IRE1 either enzymatically or nonenzymatically, with significant implications for IRE1s biological role and therapeutic targeting.

cancer biology↗

bioRxiv2023

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthase (FPGS), which normally promotes intracellular accumulation and activity of both natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. In this study, we characterize a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase (DHFR) and downstream one-carbon metabolism. Contrary to methotrexate, however, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folate concentrations for interaction with DHFR. Indeed, we show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1-induced growth inhibition, while FPGS-high CRC organoids are more sensitive to methotrexate. Our results thus argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, employing a vulnerability created by polyglutamylation deficiency.

biochemistry↗

bioRxiv2023

Nicotinic acetylcholine receptor signaling maintains epithelial barrier integrity

Disruption of epithelial barriers is a common disease manifestation in chronic degenerative diseases of the airways, lung and intestine. Extensive human genetic studies have identified risk loci in such diseases, including in chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (IBD). The genes associated with these loci have not fully been determined, and functional characterization of such genes requires extensive studies in model organisms. Here, we report the results of a screen in Drosophila melanogaster that allowed for rapid identification, validation and prioritization of COPD risk genes that were selected based on risk loci identified in human genome-wide association studies (GWAS) studies. Using intestinal barrier dysfunction in flies as a readout, our results validate the impact of candidate gene perturbations on epithelial barrier function in 56% of the cases, resulting in a prioritized target gene list. We further report the functional characterization in flies of one family of these genes, encoding for nicotinic acetylcholine receptor subunits (nAchR). We find that nAchR signaling in enterocytes of the fly gut promotes epithelial barrier function and epithelial homeostasis by regulating the production of the peritrophic matrix. Our findings identify COPD associated genes critical for epithelial barrier maintenance, and provide insight into the role of epithelial nAchR signaling for homeostasis.

genetics↗

bioRxiv2021

Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from SSc patients deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This can be mimicked in healthy fibroblasts stimulated by soluble factors that drive inflammation and fibrosis in SSc and is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

immunology↗

bioRxiv2021

Morpho-functional characterization of the endo-lysosomal system by high-throughput correlative light-electron microscopy

Rab5, EEA1 and APPL1 are frequently used in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as marker for late endosomes and lysosomes. However, since these proteins localize poorly in immuno-electron microscopy, systematic studies on their ultrastructural distributions are lacking. Here we address this gap by presenting a quantitative, high-throughput, on-section correlative light-electron microscopy (CLEM) approach using the sensitivity of fluorescence microscopy to infer label to hundreds of organelles classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 co-localizes with Rab5 on especially early endosomes, but unexpectedly also labels Rab5-negative late endosomes and even lysosomes. APPL1 is restricted to small Rab5-positive, vesicular profiles without any visible content or ultrastructural marks. Rab7 primarily labels late endosomes and lysosomes. Our studies reveal the first ultrastructural distribution of key endosomal proteins at their endogenous levels and introduce CLEM as sensitive alternative for quantitative immuno-EM.

cell biology↗

bioRxiv2021

Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.

cell biology↗

bioRxiv2021

Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (<100nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) data sets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated in a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume-EM, and obviates the need for post correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D. SignificanceWe have developed a correlative imaging pipeline to (i) correlate 3D-FM to volume-EM data with high precision, directly bridging the FM and EM resolutions (ii) achieve high-throughput volume-CLEM by targeted EM imaging of a single organelle sized region-of-interest, pre-identified by FM (iii) link live-cell fluorescence imaging of cultured mammalian cells to high-throughput volume-CLEM (iv) quantitatively study structure-function relations at subcellular scale (v) link rare (e.g. membrane contact sites) and transient (e.g. organelle interactions) cellular events to 3D ultrastructure. The targeted volume-CLEM pipeline provides a unique prospect for multi-modal correlative intracellular analysis combining dynamic interaction (live-cell imaging), functional state (live-cell imaging), molecular localization (FM), and 3D-ultrastructure (FIB.SEM) at nanometer scale.

cell biology↗

bioRxiv2021

The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes

The signaling network of the unfolded protein response (UPR) adjusts the protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1, spans the ER membrane and activates through oligomerization. IRE1 oligomers accumulate in dynamic foci. We determined the in-situ structure of IRE1 foci by cryogenic correlated light and electron microscopy (cryo-CLEM), combined with electron cryo-tomography (cryo-ET) and complementary immuno-electron microscopy. IRE1 oligomers localize to a network of narrow anastomosing ER tubes (diameter ~28 nm) with complex branching. The lumen of the tubes contains protein filaments, likely composed of linear arrays of IRE1 lumenal domain dimers, arranged in two intertwined, left-handed helices. Our findings define a previously unrecognized ER subdomain and suggest positive feedback in IRE1 signaling.

cell biology↗

bioRxiv2020

Nutrient signaling pathways regulate amyloid clearance and synaptic loss in Alzheimer's disease

Extra-cellular accumulation of Amyloid-{beta} (A{beta}) plaques is causatively associated with Alzheimers disease (AD). However, mechanisms that mediate the pre-pathological state of amyloid plaque formation remain elusive. Here, using paired RNAi and kinase inhibitor screens, we discovered that AKT-mediated insulin/nutrient signaling suppresses lysosomal clearance of A{beta} and promotes amyloid formation. This mechanism is cell-autonomous and functions in multiple systems, including iPSC-derived human neurons and in vivo. Nutrient signaling regulates amyloid formation via distinct lysosomal functional mechanisms, while enhanced amino acid signaling promotes amyloid formation by transcriptionally suppressing lysosome biogenesis, and high intracellular cholesterol levels suppress lysosomal clearance of amyloid by increasing the number of non-functional lysosomes. The nutrient signaling pathway, present in both neurons and microglia, regulates lysosomal clearance of amyloid and microglia mediated synapse loss, both in vitro and in vivo. Clinically, older hyperlipidemic patients showed less synapse loss through microglia and performed better in cognitive tests. Thus, our results reveal a bi-partite cellular quality control system regulated by the insulinnutrient signaling that in neurons regulates A{beta} peptide clearance and in microglia regulates synaptic loss, both processes causally associated with AD. Our results also caution against reducing amyloid through such processes as this might also result in synapse loss.

neuroscience↗

bioRxiv2020

Aquaporin-3 regulates endosome-to-cytosol transfer via lipid peroxidation for cross presentation

Antigen cross presentation, whereby exogenous antigens are presented by MHC class I molecules to CD8+ T cells, is essential for generating adaptive immunity to pathogens and tumor cells (1). Following endocytosis, it is widely understood that protein antigens must be transferred from endosomes to the cytosol where they are subject to ubiquitination and proteasome degradation prior to being translocated into the endoplasmic reticulum (ER), or possibly endosomes, via the TAP1/TAP2 complex (2, 3). Revealing how antigens egress from endocytic organelles (endosome-to-cytosol transfer, ECT), however, has proved vexing. Here, we used two independent screens to identify the hydrogen peroxide-transporting channel aquaporin-3 (AQP3) as a regulator of ECT. AQP3 overexpression increased ECT, whereas AQP3 knockout or knockdown decreased ECT. Mechanistically, AQP3 appears to be important for hydrogen peroxide entry into the endosomal lumen where it affects lipid peroxidation and subsequent antigen release. AQP3-mediated regulation of ECT was functionally significant, as AQP3 modulation had a direct impact on the efficiency of antigen cross presentation in vitro. Finally, AQP3-/- mice exhibited a reduced ability to mount an anti-viral response and cross present exogenous extended peptide. Together, these results indicate that the AQP3-mediated transport of hydrogen peroxide can regulate endosomal lipid peroxidation and suggest that compromised membrane integrity and coordinated release of endosomal cargo is a likely mechanism for ECT.

immunology↗