Tauopathies are a class of neurodegenerative diseases characterized by the accumulation of hyperphosphorylated, misfolded and aggregated Tau proteins and by dysfunctions in the autophagy-lysosome system. Whether the latter are a cause or a consequence of the former is unclear. The answer may come from a recent study by Mirfakhar et al. Using human iPSC-derived neurons harboring the MAPT p.R406W mutation in Tau, they were able to show that pathogenic Tau is able to broadly impair lysosomal function ahead of Tau accumulation. They also show that the degradative function of lysosomes, but not their motility, can be restored through pharmacological activation of autophagy, leading to reduced Tau levels. This work opens new therapeutic opportunities to eliminate early-on pathological misfolded Tau proteins before they can engage in a vicious cycle of aggregation, amplification and propagation.
Clathrin-independent endocytosis (CIE) comprises a diverse repertoire of internalization mechanisms that operate in parallel with clathrin-mediated endocytosis. While these mechanisms share certain characteristics, they are governed by distinct molecular principles and regulatory logic. In this review, we provide an overview of CIE mechanisms, with a particular focus on the functional specialization of individual routes. We trace the historical emergence of CIE concepts and highlight how bacterial toxins and viruses have served as pivotal discovery tools, revealing lipid-driven and lectin-mediated mechanisms of membrane bending and internalization. Furthermore, we delineate the molecular frameworks of major CIE mechanisms and discuss experimental strategies for their dissection. We emphasize how the selection of a specific endocytic route dictates cargo fate, directing receptors and adhesion molecules toward recycling, retrograde trafficking, or degradation to modulate signaling amplitude, polarity, migration and epithelial barrier functions. Finally, we offer an evolutionary perspective on "coatless" CIE-like mechanisms.
Ferroptosis has emerged as an important regulator of skeletal homeostasis, yet its role in osteoporosis (OP) remains incompletely understood. Accumulating evidence indicates that ferroptosis is not a uniform cell death program within bone, but rather a cell type-specific fate governed by distinct iron-handling capacities, redox buffering systems, and microenvironmental cues. This heterogeneity provides a new lens through which the complex pathogenesis of OP can be reinterpreted. In this review, we integrate recent advances to delineate ferroptosis-regulatory networks across major bone-resident cell populations, including osteoblasts, osteoclasts, osteocytes, and bone marrow mesenchymal stem cells (BMSCs). We highlight how ferroptosis suppresses osteogenic function in osteoblasts, amplifies differentiation and inflammatory signaling in osteoclasts, acts as an early vulnerability node in osteocytes, and reshapes lineage commitment in BMSCs. Importantly, ferroptosis in these cells is dynamically modulated by intercellular communication and niche-derived metabolic and mechanical signals. Building on this cell type-resolved framework, we propose that OP represents a disorder of multicellular ferroptotic dysregulation within the bone microenvironment rather than a simple imbalance of formation and resorption. Finally, we discuss translational implications, emphasizing ferroptosis-informed therapeutic strategies, including redox reprogramming, iron flux modulation, extracellular vesicle-based approaches, and microenvironment-responsive biomaterials. This integrative perspective provides a conceptual foundation for precision interventions targeting skeletal fragility across aging and disease contexts.
Recent work by Mao and colleagues identifies a distinct class of small extracellular vesicles, termed autophagic extracellular vesicles (AEVs), generated from amphisomes upon autophagy induction. In this commentary, we discuss how this study provides important mechanistic insight into the coupling between autophagy and secretion. AEVs are molecularly and functionally distinct from canonical exosomes, being enriched in autophagy-related components such as LC3 and p62, and dependent on core ATG machinery for their biogenesis. Notably, their secretion is enhanced by autophagy induction and contributes to intercellular communication, particularly in the context of viral infection. These findings position amphisomes as critical sorting hubs that direct cargo toward either degradation or secretion, thereby integrating autophagic and endolysosomal pathways. We further highlight how these results intersect with prior evidence implicating SNARE-dependent mechanisms, including VAMP7 and stress-responsive regulators such as GRASP55, in unconventional secretion. Finally, we discuss key unresolved questions, particularly the mechanisms underlying the generation of small intraluminal vesicles within amphisomes and the role of ESCRT machinery in this process. Overall, the identification of AEVs adds a new layer of complexity to extracellular vesicle biology and opens new avenues for understanding how autophagy contributes to intercellular signaling in health and disease.
Just as Antelope Canyon transforms familiar rock into an unexpected hidden landscape, the topological inversion of Src reveals an unforeseen extracellular facet of kinase signaling in cancer. Antelope Canyon, Arizona, USA, credit Julie Gavard.
Eps15 homology domain-containing proteins comprise a conserved family of membrane-remodeling ATPases that regulate endocytic trafficking, membrane fission, receptor recycling, primary ciliogenesis and membrane dynamics across eukaryotes. Since the initial identification of EHD1 and its Caenorhabditis elegans homolog RME-1 as regulators of endocytic recycling, research over the past quarter century has expanded the functional scope of EHD proteins far beyond classical receptor return to the plasma membrane. In mammals, EHD1, EHD2, EHD3, and EHD4 occupy overlapping but distinct cellular locations and regulate diverse processes including tubular recycling endosome fission, caveolae stabilization, primary ciliogenesis, centrosome duplication, cytokinesis, mitochondrial homeostasis, lipid droplet biology, and lipophagy. These cellular functions are supported by extensive studies in cultured cells and animal models, including mice, zebrafish, flies, worms, and plants, highlighting both conserved and specialized roles for EHD orthologs. EHD dysfunction has also been associated with a broad range of human diseases, including metabolic and cardiovascular disorders, inflammatory and infectious disease, neurologic conditions, cancer, and ciliopathies. Although many disease links remain correlative or model-based, the recent identification of an EHD1 founder mutation causing proteinuria, hearing loss, and polycystic kidney disease provides direct genetic evidence connecting EHD dysfunction to human pathology. This review summarizes 25 years of EHD research, emphasizing how EHD proteins coordinate membrane trafficking, organelle remodeling, and disease-relevant cellular physiology.
Clathrin-mediated endocytosis (CME) relies on the dynamic assembly and remodeling of clathrin coats to drive membrane curvature and vesicle formation at the plasma membrane. Although live-cell fluorescence microscopy has provided critical insights into the timing and molecular composition of endocytic events, directly linking the nanoscale lateral organization of clathrin coats to their three-dimensional progression in real time has remained challenging. Structural approaches such as electron microscopy provide detailed snapshots of clathrin architecture but are inherently static, whereas axial TIRF-based methods report membrane-proximal position with limited lateral resolution. Here, we introduce variable-angle total internal reflection fluorescence structured illumination microscopy (vaTIRF-SIM), a live-cell imaging strategy that integrates lateral super-resolution with dynamic axial sensitivity near the plasma membrane. By combining TIRF-SIM with controlled variation of the evanescent field penetration depth, vaTIRF-SIM enables simultaneous visualization of clathrin coat architecture and relative axial displacement with high spatial and temporal resolution. Applying this approach to de novo clathrin-coated pits reveals coordinated lateral growth and progressive axial advancement from early stages of pit formation through maturation, consistent with early curvature generation that intensifies over time. Extending this analysis to clathrin plaques uncovers two distinct plaque-associated endocytic behaviors: slowly maturing pits that originate at plaque peripheries and progress similarly to de novo pits and rapid plaque subdomain internalization events marked by accelerated axial progression. Together, these results establish vaTIRF-SIM as an approach that, for the first time, enables direct real-time coupling of nanoscale clathrin coat organization with axial progression during CME in living cells, demonstrated here in genome-edited SUM-159 cells expressing AP2-EGFP from the endogenous locus.
Clathrin-mediated endocytosis (CME) is an essential and conserved process in all eukaryotes. Plants, however, have evolutionarily retained remarkable endocytic machinery that distinguishes them from yeast and animals. In plants, CME execution involves the cooperative action of the heterotetrameric Adaptor protein 2 complex (AP-2) and the hetero-octameric TPLATE complex (TPC). Plants furthermore rely on the concerted action of two types of dynamin-related proteins during the final stages of clathrin-coated vesicle formation and do not use the actin cytoskeleton to generate the force to initiate membrane bending. Plants also rely more on phosphorylation and ubiquitination, rather than on canonical linear motifs, to integrate cargo sorting with endocytic dynamics. This review covers the entire process of CME in plants. It emphasizes recent advances in our mechanistic understanding of the process and highlights similarities and differences between plants and other model systems.
Fluorescent proteins that are fused to nuclear localization signals (NLSs) or nucleolar localization signals (NoLSs) are commonly used to investigate the accumulation of proteins within the nucleus and nucleolus. These reporters allow for the in vivo evaluation of signal sequence function, independent of the surrounding protein context. Here, we present a standardized protocol for measuring nuclear and nucleolar accumulation using NLS- and NoLS-fusion reporters. The workflow integrates plasmid construction, transfection, expression control, imaging and computational analysis to minimize artifacts and enhance reproducibility. This framework provides a robust basis for the reliable quantification of NLS- and NoLS-mediated localization and allows for the comparison of nuclear transport mechanisms across studies.
Erv14 is a cargo receptor of COPII vesicles, which is necessary for the efficient trafficking of various membrane proteins. In this work, we demonstrate that the deletion of the ERV14 gene impacts various physiological functions of the vacuole. Compared to the wild-type cells, cells lacking the ERV14 gene exhibited higher vacuolar pH and altered vacuolar morphology with increased fragmentation. In addition, erv14Δ cells exhibit a thinner cell wall and an impaired process of endocytosis. We also found the importance of ERV14 for cells to overcome environmental stresses, such as neutral external pH, increased zinc and calcium concentrations, and high temperature. Furthermore, comparing gene expression, proteome analysis, and structural modeling revealed new interactions between Erv14 and several vacuolar proteins, including subunits of V-ATPase and other proteins involved in carbon metabolism.
Oxytocin plays a critical role in social behavior and maternal physiology, yet the intracellular dynamics of oxytocin-containing vesicles in neurons remain poorly characterized. Here, we combine experimental data from live cell imaging of oxytocin-containing compartments with computational analysis to investigate their mobility within hypothalamic neurons. Using machine learning-based trajectory classification, we reveal that the majority of oxytocin compartments exhibit subdiffusive motion, suggesting constraints imposed by the complex intracellular environment. This behavior likely reflects interactions with cytoskeletal structures, vesicle maturation states, or localized functional demands. Our findings provide new insights into the intracellular trafficking of neuropeptides and highlight the utility of data-driven approaches for uncovering mechanisms of neurophysiological relevance.
Cell-matrix adhesion regulates Golgi organization along microtubules (MTs), though how it couples with motor-driven Golgi positioning and function remains unclear. Our earlier work showed that loss of adhesion preferentially reduces Arf1 activation at the trans-Golgi (relative to cis-Golgi), possibly affecting dynein recruitment and could cause greater trans-Golgi disorganization along MTs. We now show that this differential Golgi disorganization leads to decreased MT acetylation, which recovers upon re-adhesion. Active Arf1 overexpression in non-adherent fibroblasts prevents Golgi disorganization and sustains MT acetylation, as also observed in T24 cancer cells. Using active-Arf1 pulldown, PLA and Co-immunoprecipitation studies, we further reveal that active Arf1 recruits KIF5B to the Golgi alongside dynein. siRNA-mediated knockdown (KD) of KIF5B or dynein disperses the Golgi into ministacks. In KD cells, loss of the juxtanuclear Golgi ribbon disrupts MT organization, which is also affected by altered MTOC positioning upon dynein KD. Notably, dispersed Golgi ministacks in single motor KDs maintain MT acetylation levels in both adherent and non-adherent cells. Dual KIF5B-dynein KD keeps the Golgi juxtanuclear but uniquely compact, supporting MT acetylation. This impairs Golgi-dependent trafficking and affects cell spreading, polarity and migration. Together, these reveal the Arf1-KIF5B-dynein crosstalk as a key regulator of adhesion-dependent Golgi organization and function.
Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular leakage by binding to cell surface receptor VEGFR2. KIF13B, a kinesin 3 family cellular motor, regulates VEGF/VEGFR2 signaling by bringing VEGFR2 to the cell surface. Regulating the trafficking of VEGFR2 could be a potential therapeutic target in pathogenesis originating from abnormal angiogenesis. However, the regulation mechanism of VEGFR2 trafficking is not fully understood. Here, we show that protein kinase D (PKD) phosphorylates KIF13B. The phosphorylation of KIF13B by PKD is required for KIF13B-VEGFR2 interaction and KIF13B-microtubule interaction. Live cell imaging using VEGFR2-mCherry revealed that the PKD inhibitor reduced the mean speed and total distance of VEGFR2 vesicular trafficking. Finally, inhibition of PKD also mitigated VEGF-induced permeability measured by trans-endothelial electrical resistance (TEER). Our results demonstrate how VEGFR2 trafficking is regulated by the phosphorylation of kinesin motor KIF13B by protein kinase PKD. We anticipate that elucidating the underlying mechanism of VEGFR2 trafficking can help progress the therapy development of the target diseases related to abnormal angiogenesis.
Misfolded proteins lacking signal sequence can be secreted into the extracellular space via an unconventional protein secretion (UcPS) process termed misfolding-associated protein secretion (MAPS), which involves HSP70 and a membrane-associated HSP70 co-chaperone named DNAJC5. Here, we show that DNAJC5 can be palmitoylated by several DHHC palmitoyl acyltransferases in human cells. Among them, DHHC11 has a modest activity toward DNAJC5, but its overexpression enriches DNAJC5 in a Golgi-associated compartment, which correlates with increased secretion. Mutagenesis studies show that a minimum DNAJC5 module (DC95) consisting of the palmitoyl acceptor-enriched cysteine string (CS) domain plus the C-terminal 62 residues and a short upstream segment is sufficient to drive palmitoylation, Golgi translocation and secretion. In contrast, removal of 5 residues from DC95 abolishes its palmitoylation, Golgi association and secretion. These findings suggest that the palmitoylation sites of DNAJC5 act with flanking sequences to control its subcellular localization and UcPS function.
Lysosomal exocytosis is a fundamental cellular process that involves the fusion of lysosomes with the plasma membrane and the release of lysosomal contents into the extracellular space. This review provides an in-depth analysis of the molecular mechanisms, physiological functions, and disease implications of lysosomal exocytosis, highlighting recent advances and novel aspects. We discuss the intricate molecular machinery that orchestrates lysosomal trafficking, docking, and fusion, as well as the critical roles of lysosomal exocytosis in maintaining cellular homeostasis, facilitating intercellular communication, and contributing to specialized cellular functions. Additionally, the review explores the complex involvement of lysosomal exocytosis in various disease states, including lysosomal storage disorders, neurodegenerative diseases, cancers, and immune system disorders, underlining its potential as a therapeutic target. By identifying current knowledge gaps and providing future research directions, this review aims to stimulate further investigation into the multifaceted nature of lysosomal exocytosis and its implications for human health and disease.
In eukaryotic cells, clathrins interact with the adaptor protein (AP) complex-2 (AP-2) to facilitate endocytosis and AP-1 to mediate secretion and trafficking between the endosome and Golgi. In Plasmodium falciparum, recent studies revealed that the Kelch domain-containing protein 13 and AP-2 participate in hemoglobin uptake via cytostomes. However, clathrins appear not to be involved in this process because they primarily associate with AP-1. To investigate the roles of clathrins in P. falciparum, we characterized the clathrin heavy chain (PfCHC), the clathrin light chain (PfCLC), and the AP-1 γ subunit (PfAP-1 γ). Extensive interactome analyses confirmed the major association of clathrins with AP-1 components alongside proteins involved in cytostome formation. Live-cell imaging and protein colocalization studies showed that PfCHC, PfCLC, and PfAP-1 γ are localized in the parasite cytoplasm, predominantly at the parasite periphery and near the cis-Golgi. Ultrastructural studies using ascorbate peroxidase 2-based electron microscopy confirmed their presence at coated vesicle-like structures at the parasite periphery and, unexpectedly, at the collars of cytostomes. Knockdown of PfCHC led to the formation of abnormally long cytostome tubes and impaired hemoglobin uptake, suggesting that clathrins are involved in cytostome processes in P. falciparum.
Copper is one of the essential micronutrients utilized as a cofactor in a wide variety of biochemical reactions of metabolic pathways, including mitochondrial respiration and innate immune response. Cellular concentration and distribution of copper is regulated by copper-specific transporters, chaperones, metallothionein proteins and amino acids. Transcription of a major copper metallothionein, CUP1 is epigenetically regulated in Saccharomyces cerevisiae. Mutations in histones dysregulate cellular copper homeostasis due to abnormal epigenetic changes and cause diseases in humans, such as cancerous growth and neurological disorders. Low or higher cellular concentration of copper is associated with disorders such as Menkes and Wilson's disease, respectively. Higher concentrations of copper cause caspase-independent cell death known as cuproptosis and haemolytic anemia. We highlighted the existing knowledge regarding the significance of epigenetics and cellular factors in the regulation of copper metabolism and copper-regulated protein trafficking. We have also proposed a few future directions to explore the role of cellular pH dynamics, stoichiometry among metals, amino acids and protein metabolism, histone modifications, autophagy and mitochondrial respiration in regulating cellular copper metabolism. Altogether, we provide a comprehensive summary of cellular factors targeting copper metabolism for dissecting the underlying complex mechanism of copper dynamics in normal physiology and diseases.
Small extracellular vesicles (sEVs) originate from endosomes formed during cellular endocytosis, have a diameter ranging from 30 to 150 nm and are membrane-bound prior to release, sEVs may also be formed by budding of the plasma membrane to form ectosomes. sEVs transport proteins, RNA, microRNAs (miRNAs), DNA, and other bioactive substances to facilitate information exchange and may function as mediators under physiological conditions. sEVs have various pathological roles, especially when produced by tumor parenchyma and stromal cells for signaling in the tumor-induced microenvironment. The vesicles are considered potential tumor markers and there are broad prospects for developing tumor therapies by inhibiting sEV production, secretion and uptake and eliminating circulating sEVs. sEVs may be modified to deliver chemotherapeutic drugs and this approach has shown promising results for tumor inhibition and improved prognosis. The current study reviews the role of sEVs in tumor development and explores the potential for tumor treatment.
In eukaryotes, protein secretion plays essential roles in intercellular communications and extracellular niche-building. Protein secretion generally requires a signal sequence that targets cargos to the canonical secretory pathway consisting of the endoplasmic reticulum (ER), the Golgi apparatus, plasma membrane, and vesicles moving between these compartments. However, cytoplasmic proteins lacking signal sequences (e.g., IL1β, Acb1, FGF2) have been detected, and many have defined functions in the extracellular space, suggesting unconventional protein secretion (UcPS) via alternative pathways. In recent years, scientists have uncovered many new UcPS paradigms, reporting a plethora of mechanisms that collectively form a new field. The inaugural Cold Spring Harbor Asia (CSHA) conference on "Molecular Mechanisms and Physiology of Unconventional Secretion" is the first meeting to bring these researchers together, providing a collegial platform for information sharing at this exciting frontier of cell biology research.
Import of proviral genome from the cytoplasm to the nucleus is a decisive step in the HIV-1 infection cycle, regulating which can decide the fate of HIV-1 infectivity. Exploring the heterogeneity in the replication potential of viruses emerging from various producer cells, we compared the infectivity dynamics of viruses produced by CD4+ T lymphocytes, a cell type that supports the HIV-1 propagation, with those from astrocytes that allow for limited viral replication. We found the viruses emerging from these two cell types not only differed in their infectivity, but also in the host proteins that get associated with these virions. We focused on Importin β1, an autonomous nuclear transport receptor, which was present in the virion fractions from CD4+ T lymphocytes but absent in those from astrocytes. Our analysis revealed that Importin β1 gets associated with the virus through interactions with HIV-1 Gag and Capsid proteins. Using Importin β1 knockout cell models, we found that virion-associated Importin β1 enhanced viral infectivity by facilitating the import of the viral Pre-integration complex (PIC) into the nucleus of an infected cell. Linking positive factors, such as Importin β1, to emerging virions can determine the viral infectivity in subsequent infection rounds, influencing disease progression.
