Tiny pH-Resolved DNA Nanospheres for Cellular Pyroptosis or Apoptosis Regulation.

The design and synthesis of nanomedicines capable of regulating programmed cell death patterns to enhance antitumor efficacy remain significant challenges in cancer therapy. In this study, we developed intelligent DNA nanospheres (NS) capable of distinguishing tiny pH changes between different endosomal compartments to regulate pyroptosis or apoptosis. These NS are self-assembled from two multifunctional DNA modules, enabling tumor targeting, acid-responsive disassembly, and photodynamic therapy (PDT) activation. By modifying the embedded i-motif sequence, the NS can be activated in early endosomes (EE) or lysosomes (Ly), producing singlet oxygen (1O2) at specific locations under laser irradiation. Our results demonstrate that EE-activated PDT induces gasdermin-E-mediated pyroptosis in tumor cells, enhancing antitumor efficacy and reducing systemic toxicity compared to Ly-activated apoptosis. This study offers new insights into the design of endosome-activated nanomedicines, advancing the biomedical applications of targeted cancer therapy.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells.

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

Cryosectioning and immunofluorescence of C. elegans reveals endogenous polyphosphate in intestinal endo-lysosomal organelles.

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C. elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment.

HIV-1 N-myristoylation-dependent hijacking of late endosomes/lysosomes to drive Gag assembly in macrophages.

Macrophages represent an important viral reservoir in HIV-1-infected individuals. Different from T cells, HIV-1 assembly in macrophages occurs at intracellular compartments termed virus-containing compartments (VCCs). Our previous research in HeLa cells - in which assembly resembles that found in infected T cells - suggested that late endosomes/lysosomes (LEL) play a role in HIV-1 trafficking towards its assembly sites. However, LEL's role during assembly at VCCs is not fully understood. Herein, we used the HIV-1-inducible cell line THP-1 GagZip as a model to study HIV-1 Gag intracellular trafficking and assembly in macrophages. We demonstrated LEL involvement at VCCs using various microscopy techniques and biochemical approaches. Live-cell imaging revealed that HIV-1 repositions LEL towards the plasma membrane and modulates their motility. We showed that Arl8bmediated LEL repositioning is not responsible of Gag trafficking to VCCs. Additionally, myristoylation inhibition by PCLX-001 decreased Gag presence on endosomes and inhibited VCCs formation, in both cell-line- and primary macrophages. In conclusion, we presented evidence supporting the idea that HIV-1 manipulates the LEL trajectory to guide Gag to VCCs in an N-myristoylation-dependent manner.

Crucial role of Snf7-3 in synaptic function and cognitive behavior revealed by conventional and conditional knockout mouse models.

Snf7-3 is a crucial component of the endosomal sorting complexes required for transport (ESCRT) pathway, playing a vital role in endolysosomal functions. To elucidate the role of Snf7-3 in vivo, we developed conventional-like and conditional Snf7-3 knockout (KO) mouse models using a "Knockout-first" strategy. Conventional-like Snf7-3 KO mice showed significantly reduced Snf7-3 mRNA expression, and older mice (25-40 weeks) exhibited impaired social recognition and increased miniature excitatory postsynaptic currents (mEPSCs). Similarly, conditional KO mice aged 8-24 weeks, with Snf7-3 specifically deleted in forebrain excitatory neurons, displayed impaired object location memory and elevated mEPSC frequency. Consistently, Snf7-3 knockdown in cultured mouse hippocampal neurons led to increased densities of pre- and postsynaptic puncta, supporting the observed increase in mEPSC frequency. In addition, enhanced dendritic complexity was observed in the medial prefrontal cortex of these mice, indicating early synaptic disturbances. Our findings underscore the critical role of Snf7-3 in maintaining normal cognitive functions and social behaviors. The observed synaptic and behavioral deficits in both conventional-like and conditional KO mice highlight the importance of Snf7-3 in specific neuronal populations, suggesting that early synaptic changes could precede more pronounced cognitive impairments.

Cell-wide arrangement of Golgi/RE units depends on the microtubule organization.

We have previously shown that Golgi stacks and recycling endosomes (REs) exist as Golgi/RE units in sea urchin embryos. In this study, we showed that Golgi/RE units were scattered throughout the cytoplasm at early developmental stages but gathered to form a "Golgi ring" surrounding the centric REs at the blastula stage. This change in the cell-wide arrangement of Golgi/RE units coincided with a dramatic change in microtubule organization from a randomly oriented cortical pattern to radial arrays under the apical plasma membrane. A single gigantic Golgi apparatus surrounding centric RE is clearly associated with the center of the radial microtubule arrays. Furthermore, we found that in some animal species belonging to different clades, Golgi stacks lack lateral connections but are likely centralized by microtubule motors. These results suggest that Golgi centralization depends on the organization of the microtubule array in addition to the lateral linking between Golgi stacks. Key words: Golgi stack, recycling endosome, Golgi-ribbon, microtubule, cilium, sea urchin, ascidian.

Analysis of the neuromuscular deficits caused by STAM1 deficiency.

The endosomal sorting complexes required for transport (ESCRT) pathway is composed of a series of protein complexes that are essential for sorting cargo through the endosome. In neurons, the ESCRT pathway is a key mediator of many cellular pathways that regulate neuronal morphogenesis as well as synaptic growth and function. The ESCRT-0 complex, consisting of HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), acts as a gate keeper to this pathway, ultimately determining the fate of the endosomal cargo. We previously showed that a single nucleotide substitution in Hgs results in structural and functional changes in the nervous system of teetering mice. To determine if these changes occurred as a function of HGS's role in the ESCRT pathway and its association with STAM1, we investigated if STAM1 deficiency also leads to a similar impairment of the nervous system. In contrast to teetering mice that die within 5 weeks of age and exhibit reduced body mass, 1-month-old Stam1 knockout mice were not visibly different from controls. However, by 3 months of age, STAM1 deficiency caused reduced muscle mass, strength, and motor performance. These changes in motor function did not correlate with either a loss in motor neuron number or abnormal myelination of peripheral nerves. Instead, the motor endplate structure was altered in the Stam1 knockout mice by 1 month of age and continued to degenerate over time, correlating with a significant reduction in muscle fiber size and increased expression of the embryonic γ acetylcholine receptor (AChR) subunit at 3 months of age. There was also a significant reduction in the levels of two presynaptic SNARE proteins, VTI1A and VAMP2, in the motor neurons of the Stam1 knockout mice. As loss of STAM1 expression replicates many of the structural changes at the motor endplates that we have previously reported with loss of HGS, these results suggest that the HGS/STAM1 complex plays a critical role in maintaining synaptic structure and function in the mammalian nervous system.

Intercellular transmission of alpha-synuclein.

An emerging theme in Parkinson's disease (PD) is the propagation of α-synuclein pathology as the disease progresses. Research involving the injection of preformed α-synuclein fibrils (PFFs) in animal models has recapitulated the pathological spread observed in PD patients. At the cellular and molecular levels, this intercellular spread requires the translocation of α-synuclein across various membrane barriers. Recent studies have identified subcellular organelles and protein machineries that facilitate these processes. In this review, we discuss the proposed pathways for α-synuclein intercellular transmission, including unconventional secretion, receptor-mediated uptake, endosome escape and nanotube-mediated transfer. In addition, we advocate for a rigorous examination of the evidence for the localization of α-synuclein in extracellular vesicles.

Breaking the final barrier: Evolution of cationic and ionizable lipid structure in lipid nanoparticles to escape the endosome.

In the past decade, nucleic acid therapies have seen a boon in development and clinical translation largely due to advances in nanotechnology that have enabled their safe and targeted delivery. Nanoparticles can protect nucleic acids from degradation by serum enzymes and can facilitate entry into cells. Still, achieving endosomal escape to allow nucleic acids to enter the cytoplasm has remained a significant barrier, where less than 5% of nanoparticles within the endo-lysosomal pathway are able to transfer their cargo to the cytosol. Lipid-based drug delivery vehicles, particularly lipid nanoparticles (LNPs), have been optimized to achieve potent endosomal escape, and thus have been the vector of choice in the clinic as demonstrated by their utilization in the COVID-19 mRNA vaccines. The success of LNPs is in large part due to the rational design of lipids that can specifically overcome endosomal barriers. In this review, we chart the evolution of lipid structure from cationic lipids to ionizable lipids, focusing on structure-function relationships, with a focus on how they relate to endosomal escape. Additionally, we examine recent advancements in ionizable lipid structure as well as discuss the future of lipid design.

Transport mechanisms between the endocytic, recycling, and biosynthetic pathways via endosomes and the trans-Golgi network.

After the endocytic and biosynthetic pathway converge, they partially share the route to the lysosome/vacuole. Similarly, the endocytic recycling and secretory pathways also partially share the route to the plasma membrane. The interaction of these transport pathways is mediated by endosomes and the trans-Golgi network (TGN), which act as sorting stations in endocytic and biosynthesis pathway, and endosomes has a bidirectional transport to and from the TGN. In mammalian cells endosomes can be largely classified as early/sorting, late, and recycling endosomes, based on their morphological features and localization of Rab family proteins, which are key factors in vesicular trafficking. However, these endosomes do not necessarily represent specific compartments that are comparable among different species. For instance, Rab5 localizes to early endosomes in mammalian cells but is widely localized to early-to-late endosomes in yeast, and to pre-vacuolar endosomes and the TGN in plant cells. The SNARE complexes are also key factors widely conserved among species and localized specifically to the endosomal membrane, but the localization of respective homologs is not necessarily consistent among species. These facts suggest that endosomes should be classified more inclusively across species. Here we reconsider the mammalian endosome system based on findings in budding yeast and other species and discuss the differences and similarities between them.

Cytokines on the way to secretion.

The activation of immune cells by pro-inflammatory or immunosuppressive stimuli is followed by the secretion of immunoregulatory cytokines which serve as messengers to activate the immune response in target cells. Although the mechanisms that control the secretion of cytokines by immune cells are not yet fully understood, several key aspects of this process have recently emerged. This review focuses on cytokine release via exocytosis and highlights the routes of cytokine trafficking leading to constitutive and regulated secretion as well as the impact of sorting receptors on this process. We discuss the involvement of cytoskeletal rearrangements in vesicular transport, secretion, and formation of immunological synapses. Finally, we describe the non-classical pathways of cytokine release that are independent of vesicular ER-Golgi transport. Instead, these pathways are based on processing by inflammasome or autophagic mechanisms. Ultimately, understanding the molecular mechanisms behind cytokine release may help to identify potential therapeutic targets in diseases associated with altered immune responses.

Interaction between ESCRT-III proteins and the yeast SERINC homolog Tms1.

The endosomal sorting complex required for transport (ESCRT)-III is involved in membrane remodeling and abscission during intraluminal vesicle (ILV) formation at endosomes. Our data now suggest that ESCRT-III function could be connected to lipid remodeling of the endosomal membrane. This notion is based on our finding that ESCRT-III proteins bind to the yeast serine incorporator (SERINC) homolog Tms1. Human SERINC3 and SERINC5 are HIV-1 restriction factors and have been shown to act as scramblases, flipping phospholipids between membrane leaflets. Due to the extraordinarily high sequence conservation between Tms1 and human SERINCs, it is likely that Tms1 is also a scramblase. While deletion of TMS1 had only a moderate effect on the sorting of multivesicular body (MVB) cargo proteins, the simultaneous deletion of a component of the Vps55/Vps68 complex led to a strong synergistic phenotype. This pronounced synergism suggests that Tms1 and Vps55/Vps68 perform a parallel function at endosomes. Vps55/Vps68 loosely resembles Tms1 in its overall structure. Thus, it is possible that Vps55/Vps68 is also a scramblase. Since both Vps55 and Tms1 physically interact with ESCRT-III proteins, we propose that the recruitment of a scramblase plays a crucial role in ESCRT-III-dependent membrane remodeling at endosomes.

Maternal choline supplementation rescues early endosome pathology in basal forebrain cholinergic neurons in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.

Individuals with DS develop Alzheimer's disease (AD) neuropathology, including endosomal-lysosomal system abnormalities and degeneration of basal forebrain cholinergic neurons (BFCNs). We investigated whether maternal choline supplementation (MCS) affects early endosome pathology within BFCNs using the Ts65Dn mouse model of DS/AD. Ts65Dn and disomic (2N) offspring from dams administered MCS were analyzed for endosomal pathology at 3-4 months or 10-12 months. Morphometric analysis of early endosome phenotype was performed on individual BFCNs using Imaris. The effects of MCS on the endosomal interactome were interrogated by relative co-expression (RCE) analysis. MCS effectively reduced age- and genotype-associated increases in early endosome number in Ts65Dn and 2N offspring, and prevented increases in early endosome size in Ts65Dn offspring. RCE revealed a loss of interactome cooperativity among endosome genes in Ts65Dn offspring that was restored by MCS. These findings demonstrate MCS rescues early endosome pathology, a driver of septohippocampal circuit dysfunction. The genotype-independent benefits of MCS on endosomal phenotype indicate translational applicability as an early-life therapy for DS as well as other neurodevelopmental/neurodegenerative disorders involving endosomal pathology.

Endolysosomal channel TMEM175 mediates antitoxin activity of DABMA.

DABMA is a chemical molecule optimized from the parent compound ABMA and exhibits broad-spectrum antipathogenic activity by modulating the host's endolysosomal and autophagic pathways. Both DABMA and ABMA inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a cellular assay, which further expands their anti-pathogen spectrum in vitro. However, their precise mechanism of action has not yet been resolved. TMEM175 is a newly characterized endolysosomal channel which plays an essential role in the homeostasis of endosomes and lysosomes as well as organelle fusion. Here, we show that DABMA increases the endosomal TMEM175 current through organelle patch clamping with an EC50 of 17.9 µm. Depletion of TMEM175 protein significantly decreases the antitoxin activity of DABMA and affects its action on acidic- and Rab7-positive endosomes as well as on endolysosomal trafficking. Thus, TMEM175 is necessary for DABMA's activity and may represent a druggable target for the development of anti-infective drugs. Moreover, DABMA, as an activator of the TMEM175 channel, may be useful for the in-depth characterization of the physiological and pathological roles of this endolysosomal channel.

Endosomal catabolism of phosphatidylinositol 4,5-bisphosphate is fundamental in building resilience against pathogens.

Endosomes are characterized by the presence of various phosphoinositides that are essential for defining the membrane properties. However, the interplay between endosomal phosphoinositides metabolism and innate immunity is yet to be fully understood. Here, our findings highlight the evolutionary continuity of RAB-10/Rab10's involvement in regulating innate immunity. Upon infection of C. elegans with P. aeruginosa, an increase in RAB-10 activity was observed in the intestine. Conversely, when RAB-10 was absent, the intestinal diacylglycerols (DAGs) decreased, and the animal's response to the pathogen was impaired. Further research revealed that UNC-16/JIP3 acts as an RAB-10 effector, facilitating the recruitment of phospholipase EGL-8 to endosomes. This leads to a decrease in endosomal PI(4,5)P2 and an elevation of DAGs, as well as the activation of the PMK-1/p38 MAPK innate immune pathway. It is noteworthy that the dimerization of UNC-16 is a prerequisite for its interaction with RAB-10(GTP) and the recruitment of EGL-8. Moreover, we ascertained that the rise in RAB-10 activity, due to infection, was attributed to the augmented expression of LET-413/Erbin, and the nuclear receptor NHR-25/NR5A1/2 was determined to be indispensable for this increase. Hence, this study illuminates the significance of endosomal PI(4,5)P2 catabolism in boosting innate immunity, and outlines an NHR-25-mediated mechanism for pathogen detection in intestinal epithelia.

Therapeutic application of extracellular vesicular EGFR isoform D as a co-drug to target squamous cell cancers with tyrosine kinase inhibitors.

Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype in vitro and in vivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.

Switching on lysosomes.

The formation of large endolysosomal structures in unfertilized eggs ensures that lysosomes remain dormant before fertilization, and then shift into clean-up mode after the egg-to-embryo transition.

CCT3/ACTN4/TFRC axis protects hepatocellular carcinoma cells from ferroptosis by inhibiting iron endocytosis.

Sorafenib is widely used in treating advanced hepatocellular carcinoma (HCC). However, its effectiveness in prolonging patient survival is limited by the development of drug resistance. To systematically investigate the resistance mechanisms of Sorafenib, an integrative analysis combining posttranslational modification (PTM) omics and CRISPR/Cas9 knockout library screening was conducted. This analysis identified ubiquitination at lysine 21 (K21) on chaperonin-containing TCP1 subunit 3 (CCT3) as being associated with Sorafenib resistance. Transcriptomic data from HCC patients treated with Sorafenib revealed that CCT3 expression was lower in responders compared to non-responders. Experimentally, inhibiting the expression of CCT3 sensitized HCC cells to Sorafenib and enhanced Sorafenib-induced ferroptosis. Additionally, CCT3 was found to interact with ACTN4, hindering the recycling of transferrin receptor protein 1 (TFRC) to the cell membrane, thus obstructing iron endocytosis. Mechanistically, the inhibition of ferroptosis by CCT3 depends on the deubiquitination of K6-linked non-degradative ubiquitination at its K21, which occurs upon Sorafenib treatment. Moreover, CCT3 knockdown enhanced the anti-tumor effects of Sorafenib in nude mice. In summary, we have identified a novel function of the chaperone protein. Targeting the CCT3/ACTN4/TFRC axis offers a promising strategy to enhance ferroptosis and overcome Sorafenib resistance in HCC.

Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27-Retromer complex.

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway.

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.