Marian Kalocsay

Cilia-derived extracellular vesicles (EVs) mediate cell-cell communication and remove signaling molecules from cilia. The Hedgehog receptor Smoothened (SMO) is selected for packaging into ciliary EVs by ubiquitin chains, but how cargo selection is coupled to EV budding remains unknown. In this work, working in mouse kidney cells, we found that the ubiquitin reader endosomal sorting complex required for transport–0 (ESCRT-0) orchestrates both cargo selection and EV biogenesis. Unlike the conventional ESCRT pathway, SMO secretion did not require the ESCRT-III membrane scission machinery. Instead, ESCRT-0 condensed at the ciliary tip, where it clustered ubiquitinated SMO to drive EV biogenesis. Disrupting ESCRT-0 condensation blocked SMO secretion, and combining forced recruitment of ESCRT-0 with optogenetic clustering of SMO was sufficient to reconstitute EV budding. Thus, ESCRT-0 condensation and cargo clustering constitute a minimal module for ESCRT-III–independent reverse-topology budding.

Receptor signaling is often framed as protein recruitment after stimulation, but whether receptor-proximal environments are organized in composition and phosphorylation state beforehand remains unclear. We used CD18-APEX2 proximity labeling and a spatial membrane reference to quantify proteins and phosphosites near leukocyte β2 integrins. At rest, receptors, adaptors and Rap GTPases were preferentially represented relative to several catalytic classes. Parent-protein correction revealed spatial phosphoform organization, with 32% of 4,564 phosphosites deviating significantly from the distribution center and 56% of proteins with multiple quantified sites showing divergence beyond measurement error. Sustained activation altered 31% of phosphosites while 95.9% of proteins remained equivalent within ±0.5 log ₂ and spatial phosphoform organization largely persisted. Most phosphorylation responses were shared across both labeling environments, whereas receptor-preferential changes were dominated by phosphosites. These findings suggest that receptor signaling can regulate pre-existing molecular states without extensive protein redistribution.

Cardiac fibrosis independently predicts adverse outcomes in heart failure (HF), yet no Food and Drug Administration–approved therapy directly targets fibrotic remodeling in the heart. To address this unmet clinical need, we used a multidimensional drug discovery pipeline centered on a human induced pluripotent stem cell (iPSC)–based platform. Through high-throughput screening, we identified CGS15943 (CGS) as the lead antifibrotic compound and validated its activity in human cardiac fibroblasts, three-dimensional engineered heart tissues, and animal models of HF. Mechanistic studies revealed an atypical adenosine receptor (AR)–dependent signaling pathway in which AR subtypes converge on Gβγ (the βγ subunits of heterotrimeric GTP-binding proteins) to activate phosphoinositide 3-kinase (PI3K)–AKT and yes-associated protein (YAP). CGS suppressed this signaling axis, thereby reducing fibrotic gene expression and fibroblast activation. These findings establish AR-driven Gβγ signaling as a potential therapeutic target for cardiac fibrosis.

Beta-arrestins (β-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of β-arr coupling, the molecular basis of GPCR/β-arr interaction has been studied primarily in monomeric family A GPCRs. Here we develop a single molecule pulldown (SiMPull) assay that reports on both relative GPCR:β-arr interaction strength and stoichiometry to uncover molecular diversity in β-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find that mGluRs couple to β-arrs with variable apparent strength and a 2:1 or 2:2 stoichiometry. Focusing on the mGluR8/β-arr1 interaction, we map the contributions of “tail” and “core” interactions with residues in the receptor C-terminal domain and intracellular loops, respectively. Combinatorial mutagenesis enables the identification of a landscape of β-arr complexes with homo- and hetero-dimeric mGluR8, including both “cis” and “trans” interactions. Finally, we use our SiMPull assay in conjunction with live cell imaging and transcriptomic analysis to reveal that mGluRs can form megacomplexes either by simultaneously binding β-arr1 and β-arr2 or β-arr and G proteins. Together, this study provides a comprehensive framework for mGluR/β-arr complex diversity, expanding the known range of GPCR/transducer coupling modes.

Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed Arl13b-TurboID mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse tissues and cell types. Comparative proteomics revealed notable tissue-specific specialization, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter receptors. Unexpectedly, several signaling and adhesion molecules localize to neuronal cilia in discrete nanodomains maintained by active retrieval mechanisms. In the mouse cortex, expansion microscopy revealed that the NMDA receptor subunit GluN1 is organized in nanodomains on neuronal ciliary membranes, which are precisely positioned to sample neurotransmitter efflux from neighboring glutamatergic synapses. These findings establish neuronal cilia as specialized extrasynaptic signaling platforms, with nanoscale organization enabling them to integrate local synaptic cues and modulate neuronal connectivity.

NANOG overexpression has been reported to reverse aging-associated decline in mesenchymal stem/stromal cell (MSC) function, but the molecular machinery engaged by NANOG in MSCs remains incompletely defined. Here, we applied APEX proximity labeling coupled with quantitative mass spectrometry to define the NANOG proximity interactome (proxeome) in human MSCs. Of 1040 quantified proteins, 828 were significantly enriched in the APEX-NANOG (H₂O₂ labeling) samples, consistent with a broad NANOG-centered neighborhood rather than a single stoichiometric complex. Enriched proteins encompass RNA-processing pathways (including splicing/RNP factors and selected m6A-related proteins), transcriptional coactivation and elongation control (Mediator and 7SK/P-TEFb regulators), chromatin repression/poising modules (Polycomb and HDAC/NuRD/CoREST/SIN3), ATP-dependent chromatin remodeling (BAF/SWI-SNF), three-dimensional genome organization and replication-coupled chromatin maintenance (CTCF/cohesin, CHAF1A, RIF1, UHRF1), and regulators of MSC identity and signal integration (Hippo/mechanotransduction and TGFβ-linked transcriptional circuits). Together, these data provide a spatial proteomic map of NANOG-associated nuclear neighborhoods in MSCs and a foundation for mechanistic hypotheses for how NANOG may stabilize stem-like programs.

Neuroinflammation is a pathological feature of neurodegenerative diseases like Alzheimer’s disease and ALS. Cytoplasmic dsRNA (cdsRNA) triggers a type-I interferon response in human neural cells, leading to their death, and is found in neurons of C9ORF72 -ALS patients. Here, we report the spatial coincidence of cdsRNA and pTDP-43 inclusions in human postmortem tissue with Alzheimer’s disease pathology, and upregulated interferon response genes in affected regions. CdsRNA also accumulates in a human TDP-43 G298S iPSC cortical neuronal model. We use cryptic exon detection as a proxy for TDP-43 mislocalization and demonstrate that FDA-approved JAK inhibitors baricitinib and ruxolitinib, which block interferon signaling, show protective effects only in brains with elevated cryptic exon expression. A CRISPR screen reveals TYK2 as a top hit, and TYK2 knockdown and the selective TYK2 inhibitor deucravacitinib rescue cdsRNA-induced toxicity. We find parallel neuroinflammatory mechanisms, dependent on TYK2 - a potential disease-modifying target - for TDP-43-associated Alzheimer’s disease and C9ORF72 -ALS.

BACKGROUND: FHF (fibroblast growth factor homologous factor) variants associate with arrhythmias. Although FHFs are best characterized as regulators of voltage-gated sodium channel (VGSC) gating, recent studies suggest broader, non-VGSC–related functions, including regulation of Cx43 (connexin 43) gap junctions and hemichannels, mechanisms that have generally been understudied or disregarded. METHODS: We assessed cardiac conduction and cardiomyocyte action potentials in mice with constitutive cardiac-specific Fgf13 ablation (c Fgf13 ^KO ) while targeting Cx43 gap junctions and hemichannels pharmacologically. We characterized FGF13 regulation of Cx43 abundance and subcellular distribution. With proximity labeling proteomics, we investigated novel candidate mechanisms underlying FGF13 regulation of Cx43. RESULTS: FGF13 ablation prolonged the QRS and QT intervals. Carbenoxolone, a Cx43 gap junction uncoupler, markedly prolonged the QRS duration, leading to conduction system block in c Fgf13 ^KO but not in wild-type mice. Optical mapping revealed markedly decreased conduction velocity during ventricular pacing. Microscopy revealed perturbed trafficking of Cx43, reduced localization in the intercalated disc, and suggested decreased membrane Cx43 but increased Cx43 hemichannels in cardiomyocytes from c Fgf13 ^KO mice. Resting membrane potential was depolarized, and action potential duration at 50% repolarization was prolonged in c Fgf13 ^KO cardiomyocytes. Both were restored toward wild-type values with Gap19 (a Cx43 hemichannel inhibitor), expression of FGF13, or expression of a mutant FGF13 incapable of binding to VGSCs, emphasizing VGSC-independent regulation by FGF13. To assess the functional impact of resting membrane potential depolarization, hearts were subjected to hypokalemia, which had no effect in wild-type hearts but fully rescued conduction velocity in c Fgf13 ^KO hearts. Proteomic analyses revealed candidate roles for FGF13 in the regulation of vesicular-mediated transport. FGF13 ablation destabilized microtubules and reduced the expression of tubulins and MAP4, the major cardiac microtubule regulator. CONCLUSIONS: FGF13 regulates microtubule-dependent trafficking and targeting of Cx43 and impacts cardiac impulse propagation via VGSC-independent mechanisms.

The small, tubulin-binding protein STMN2 is highly expressed in neurons and is implicated in amyotrophic lateral sclerosis. STMN2 degrades rapidly and accumulates at axotomy sites, suggesting fast turnover is crucial for its neuroprotective function. We show that STMN2 was primarily degraded by the ubiquitin–proteasome system. Its membrane-targeting N-terminal domain promoted fast turnover, whereas its tubulin-binding domain promoted stabilization. Proximity labeling and imaging showed that tubulin binding reduced STMN2 targeting to trans-Golgi network membranes. Pull-down assays showed that tubulin binds preferentially to soluble over membrane-bound STMN2. Our observations suggest that STMN2 interconverts between a soluble, tubulin-bound form and a membrane-bound, tubulin-free form, and is rapidly degraded when released from both membranes and tubulin. We propose that tubulin binding sequesters and stabilizes STMN2, while its neuroprotective function involves an unknown membrane activity.

Lysosomes are essential organelles for cellular homeostasis and signaling, with dysfunction linked to neurological disorders, lysosomal storage diseases, and cancer. While proteomics has advanced our understanding of lysosomal composition, the structural characterization of lysosomal membrane proteins in their native environment remains a significant challenge. Here, we developed a cryo electron tomography workflow to visualize lysosomal membrane proteins within intact, native lysosomal membranes. We isolated endolysosomes by independently targeting two lysosomal membrane proteins, transient receptor potential mucolipin 1 and transmembrane protein 192, enriching organelles that exhibited the expected morphology and proteomic composition of the endolysosomal system. Sub-tomogram averaging enabled the structural refinement of key membrane and membrane-associated proteins, including V-ATPase, Flotillin, and Clathrin, directly within the lysosomal membrane, revealing their heterogeneous distribution across endolysosomal organelles. By integrating proteomics with structural biology, our workflow establishes a powerful platform for studying lysosomal membrane protein function in health and disease, paving the way for future discoveries in membrane-associated lysosomal mechanisms.