Caveolae-mediated Endocytosis Research Articles

Spleen Targeting Nucleic Acid Delivery Vector Based on Metal-Organic Frameworks.

Nucleic acids have attracted increasing attention as drugs due to their fascinating advantages, such as long-term efficacy and ease of preparation compared to proteins. The nucleic acid therapy relies heavily on delivery vectors, which can prevent the degradation of nucleic acids while assisting them in cellular internalization. However, commonly used nonviral vector liposomes easily accumulate in the liver, which can limit their application in extrahepatic diseases. Herein, a potential spleen targeting vector for nucleic acids is developed based on the metal-organic frameworks. The plasmids are encapsulated inside the nanoscale zeolitic imidazolate framework (ZIF) via coprecipitation. The co-encapsulation of the cationic polymer poly(ether imide) (PEI) and the stabilizer polyvinylpyrrolidone (PVP) can significantly improve particle dispersion and stability. The prepared nanoparticles allow efficient transfection in vitro, mainly through clathrin-mediated and caveolae-mediated endocytosis. The biodistribution in mice shows that 46% of the nanoparticles accumulate in the spleen, which is much higher than that of the liposomes. The vector can successfully deliver plasmids to extrahepatic organs for protein synthesis and even induce an immune response. The elaborate ZIF-based nanoparticle may offer a new route for extrahepatic, especially spleen targeting delivery for the nucleic acids.

Nuclear translocation of plasma membrane protein ADCY7 potentiates T cell-mediated antitumour immunity in HCC

BackgroundThe potency of T cell-mediated responses is a determinant of immunotherapy effectiveness in treating malignancies; however, the clinical efficacy of T-cell therapies has been limited in hepatocellular carcinoma (HCC) owing...

Nonviral genetic manipulation system with microenvironmental cascade sensitivity for the treatment of TNBC-associated metastatic bone tumor

Gene therapy is an efficacious therapeutic modality for triple negative breast cancer (TNBC). However, the practicable applicability is strictly confined in TNBC-associated metastatic bone tumor due to the low blood flow and poor accumulation in bone tissue, as well as the vicious cycle of mutual promotion between tumor growth and bone erosion. To address those problems, a versatile nonviral genetic manipulation system PSMCA/miR-34a, with caveolae-activated endocytosis and microenvironmental cascade-responsive release profile was exploited. In vitro and in vivo experiments confirmed that PSMCA/miR-34a not only possessed bone-targeting properties to accumulate at bone tumor tissue, also could respond to the bone tumor microacidic environment to release alendronate to inhibit the bone erosion. After internalized into cells mainly through caveolae-mediated endocytosis, it was spurred to disassemble for controlling release the miR-34a by high intracellular GSH environment, generating an effective anti-tumor effect. Moreover, due to the synergistic effect, PSMCA/miR-34a could breakdown the vicious cycle between bone destruction and tumor growth in the tumor microenvironment, showing high potential in the clinical gene therapy for TNBC-associated metastatic bone tumor.

PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy.

Polyethylene glycol (PEG) is a common surface modification for lipid nanoparticles (LNPs) to improve their stability and in vivo circulation time. However, the impact of PEGylation on LNP cellular uptake remains poorly understood. To tackle this issue, we systematically compared plain and PEGylated LNPs by combining dynamic light scattering, electrophoretic light scattering, and synchrotron small-angle X-ray scattering (SAXS) that unveils a striking similarity in size and core structure but a significant reduction in surface charge. Upon administration to human embryonic kidney (HEK 293) cells, plain and PEGylated LNPs were internalized through different endocytic routes, as revealed by spatiotemporal correlation spectroscopy. An imaging-derived mean square displacement (iMSD) analysis shows that PEGylated LNPs exhibit a significantly stronger preference for caveolae-mediated endocytosis (CAV) and clathrin-mediated endocytosis (CME) pathways compared to plain LNPs, with these latter being better tailored to MCR-dependent internalization and trafficking. This suggests that PEG plays a crucial role in directing LNPs toward specific cellular uptake routes. Further studies should explore how PEG-mediated endocytosis impacts intracellular trafficking and ultimately translates to therapeutic efficacy, guiding the design of next-generation LNP delivery systems.

Borneol and lactoferrin dual-modified crocetin-loaded nanoliposomes enhance neuroprotection in HT22 cells and brain targeting in mice

Crocetin (CCT), a natural bioactive compound extracted and purified from the traditional Chinese medicinal herb saffron, has been shown to play a role in neurodegenerative diseases, particularly depression. However, due to challenges with solubility, targeting, and bioavailability, formulation development and clinical use of CCT are severely limited. In this study, we used the emulsification-reverse volatilization method to prepare CCT-loaded nanoliposomes (CN). We further developed a borneol (Bor) and lactoferrin (Lf) dual-modified CCT-loaded nanoliposome (BLCN) for brain-targeted delivery of CCT. The results of transmission electron microscope (TEM) and particle size analysis indicated that the size of BLCN (∼140 nm) was suitable for transcellular transport across olfactory axons (∼200 nm), potentially paving a direct path to the brain. Studies on lipid solubility, micropolarity, and hydrophobicity showed that BLCN had a relatively high Lf grafting rate (81.11 ± 1.33 %) and CCT entrapment efficiency (83.60 ± 1.04 %) compared to other liposomes, likely due to Bor improving the lipid solubility of Lf, and the combination promoting the orderly arrangement of liposome membrane molecules. Microplate reader and fluorescence microscopy analysis showed that BLCN efficiently promoted the endocytosis of fluorescent coumarin 6 into HT22 cells with a maximal fluorescence intensity of (13.48 ± 0.80 %), which was significantly higher than that of CCT (5.73 ± 1.17 %) and CN (12.13 ± 1.01 %). BLCN also exhibited sustained function, remaining effective for more than 12 h after reaching a peak at 1 h in cells, while CN showed a significant decrease after 4 h. The uptake mechanisms of BLCN in HT22 cells mainly involve energy-dependent, caveolae-mediated, and microtubule-mediated endocytosis, as well as micropinocytosis. Furthermore, BLCN displayed a significant neuroprotective effect on HT22 cells in glutamate-, corticosterone-, and H2O2-induced models. Tissue fluorescence image analysis of mice showed that BLCN exhibited substantial retention of fluorescent DiR in the brain after nasal administration for 12 h. These findings suggest that CCT has the potential for cellular uptake, neuroprotection, and targeted delivery to the brain following intranasal administration when encapsulated in Bor and Lf dual-modified nanoliposomes.

NLC Delivery of EGFP Plasmid to TM4 Cell Nuclei for Targeted Gene Therapy.

This study evaluated whether a nanostructured lipid carrier (NLC) delivery system could safely and accurately deliver nucleic acids to the cell nucleus using the enhanced green fluorescent protein (EGFP)-C1 plasmid model. The NLC was formulated using the emulsification method and equipped for cationic lipid-mediated transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which interacts electrostatically with nucleic acid. The NLC attributes, including size, polydispersity index, and zeta potential, were assessed by dynamic light scattering (DLS). The morphological structure was analyzed using transmission electron microscopy. Entrapment efficiency was evaluated by a direct method. Cellular uptake mechanisms of pEGFP-C1-NLC and the ability of pEGFP-C1 to penetrate the nucleus of TM4 cells to express EGFP were observed using confocal microscopy. pEGFP-C1-NLC exhibited particle sizes in the range 56-88 nm with a particle charge range of -6.0 to+1.3 mV. The polydispersity index<0.5 showed good size uniformity, and entrapment efficiency of pEGFP-C1in the NLC was 92.06±2.295%. The NLC formulation was internalized predominantly via caveolae-mediated endocytosis, as indicated by EGFP expression following successful delivery of pEGFP by the NLC into the cells. NLC formulation could deliver genetic material to the nucleus and could be considered a gene therapy candidate for spermatogenesis.

Charge-switchable cell-penetrating peptides for rerouting nanoparticles to glioblastoma treatment

Glioblastoma (GB) is one of the most lethal types of neoplasms with unique anatomic, physiologic, and pathologic features that usually persist after exposure to standard therapeutic modalities. It is biologically aggressive, and the existence of the blood-brain barrier (BBB) limits the efficacy of standard therapies. In this work, we hypothesize the potential of surface-functionalized ultra-small nanostructured lipid carriers (usNLCs) with charge-switchable cell-penetrating peptides (CPPs) to overcome this biological barrier and improve targeted delivery to brain tumor tissues. The big question is: what is the potential of CPPs in directing nanoparticles toward brain tumor tissue? To answer this question, the usNLCs were functionalized with distinct biomolecules [five CPPs, c(RGDfK) and transferrin, Tf] through electrostatic interaction and its ability as a targeting approach to BBB (HBMEC) and glioma cells (U87 cells) evaluated in terms of physicochemical properties, cellular uptake, permeability in a 2D-BBB model, and tumor growth inhibition. Monte Carlo simulations elucidated CPP adsorption patterns. The permeability studies revealed that targeted usNLCs, especially usNLCsTf and usNLCsCPP4, exhibited an increased permeability coefficient compared to the non-targeted usNLCs. Functionalized usNLCs evidenced enhanced uptake in BBB cells, with smaller CPPs showing higher internalization (CPP1 and CPP2). Similarly, functionalized usNLCs exhibited more significant cytotoxicity in glioma cells, with specific CPPs promoting favorable internalization. Analysis of the endocytic pathway indicated that usNLCsCPPs were mainly internalized by direct translocation and caveolae-mediated endocytosis. Optimal usNLCs with dual targeting capabilities to both BBB and GB cells provide a promising therapeutic strategy for GB.

Intestinal lymphatic transport of Smilax china L. pectic polysaccharide via Peyer's patches and its uptake and transport mechanisms in mononuclear phagocytes

Recently, the intestinal lymphatic transport based on Peyer's patches (PPs) is emerging as a promising absorption pathway for natural polysaccharides. Herein, the aim of this study is to investigate the PP-based oral absorption of a pectic polysaccharide from Smilax china L. (SCLP), as well as its uptake and transport mechanisms in related immune cells. Taking advantages of the traceability of fluorescently labeled SCLP, we confirmed that SCLP could be absorbed into PPs and captured by their mononuclear phagocytes (dendritic cells and macrophages) following oral administration. Subsequently, the systematic in vitro study suggested that the endocytic mechanisms of SCLP by model mononuclear phagocytes (BMDCs and RAW264.7 cells) mainly involved caveolae-mediated endocytosis, macropinocytosis and phagocytosis. More importantly, SCLP directly binds and interacts with toll-like receptor 2 (TLR2) and galectin 3 (Gal-3) receptor, and was taken up by mononuclear phagocytes in receptor-mediated manner. After internalization, SCLP was intracellularly transported primarily through endolysosomal pathway and ultimately localized in lysosomes. In summary, this work reveals novel information and perspectives about the in vivo fate of SCLP, which will contribute to further research and utilization of SCLP and other pectic polysaccharides.

Arachidonic Acid Metabolism by Cyp4A Modulates Caveolae-Mediated Transcytotic LPS Transport in Mouse Jejunum

Arachidonic acid (AA) is metabolized not only by cyclooxygenases and lipoxygenases, but also by cytochrome P-450 4A family (Cyp4A) which is an androgen-driven enzyme that produces 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is an endogenous ligand for GPR75, which is highly associated with pathogenesis of obesity. Since metabolic endotoxemia is implicated in the induction of obesity, and the gut lumen is the primary source of endotoxin (lipopolysaccharides [LPS]), we hypothesized that AA metabolism may affect LPS entry through the small intestine. Furthermore, we have found that increased LPS transport during long-chain fatty acid (LCFA) absorption is abolished in caveolin-1 (Cav1) knockout (KO) jejunum, suggesting the involvement of caveolae-mediated endocytosis. We thus examined the effects of AA on LPS transport in murine jejunum. FITC-LPS (10 μg/ml) or FD4 (0.1 mM) was applied to the mucosal (m) bath of Ussing chambered muscle-stripped jejunum from male C57Bl6 wild type (WT) and Cav1 KO mice with measurement of the serosal (s) appearance of FITC-LPS or FD4 and transepithelial electrical resistance (TER). We examined the effects of the luminal application (10 mM) of oleic acid (OA), AA, eicosapentaenoic acid (EPA), or docosahexaenoic (DHA) with taurocholic acid (0.1 mM) on these parameters with or without the lipid raft inhibitor methyl-β-cyclodextrin (MβCD, 0.1 mM) or Cyp4A inhibitor HET0016 (10 μM). In another study, male and female WT and Cav1 KO mice were fed a standard diet (SD), or a diet enriched in saturated LCFA fat and sugar (LCD) for 6 wks. Luminal addition of OA and AA, but not EPA nor DHA, increased LPS m-to-s movement in WT jejunal mucosa. AA also increased FD4 m-to-s movement with no effect on TER. AA-induced LPS and FD4 movements were inhibited by luminal application of MβCD or HET0016, suggesting the involvement of lipid rafts and Cyp4A activity. Luminal AA had no effect on LPS and FD4 m-to-s movement in KO jejunum. LPS and FD4 m-to-s movement were also increased by luminal application of 20-HETE (0.3 and 1 μM). Immunostaining revealed that GPR75 was expressed on the plasma membranes of jejunal villous but not crypt cells. LCD induced obesity in WT, not in KO, with increased blood glucose levels, perigonadal fat weight, and liver weight, with changes more evident in male WT than in female WT. LCD increased LPS m-to-s movement in WT male jejunum, not in KO jejunum, which was inhibited by luminal addition of MβCD or HET0016. These results suggest that luminal AA increases 20-HETE production by Cyp4A, then activates Cav1-mediated endocytosis which in turn enhances LPS transport in the small intestine, possibly via GPR75 activation. The AA-Cyp4A-20-HETE-GPR75 system may be a new therapeutic target for metabolic endotoxemia associated with diet-induced obesity, especially in males. VA Merit Review and DoD grants. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High Fat Diet-Induced Constitutive Caveolin-1 Activation in Mouse Jejunum Promotes LPS Transport with Consequent Metabolic Syndrome

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported from the gut lumen to the portal vein (PV) in the presence of luminal long-chain fatty acid (LCFA) via CD36- and lipid raft-mediated pathways. Furthermore, LCFA-induced LPS transport is abolished in caveolin-1 (Cav1) knockout (KO) jejunum, suggesting the involvement of caveolae-mediated endocytosis. Here, we examined the effects of diets of varying FA composition on the development of the metabolic syndrome (MetS) and on LPS transport in murine jejunum. Male and female C57Bl6 wild type (WT) and Cav1 KO mice were fed a standard high CHO, high protein, low fat diet (SD), or one of 3 high fat-high sugar diets (34% sucrose, 42% fat) that were enriched in saturated LCFA (LCD), medium-chain FA (MCD) and polyunsaturated FA (PUD) for 6 wks. FITC-LPS was added to the mucosal (m) bath of Ussing-chambered muscle-stripped jejunum. Serosal (s) appearance of FITC-LPS was measured with or without luminal addition of oleic acid (OA, 10 mM)/taurocholic acid (TCA, 0.1 mM). FD4 m-to-s permeability and transepithelial electrical resistance (TER) were also measured. PV LPS levels were measured by a limulus amœbocyte lysate test kit. LCD-induced MetS (increased body weight, blood glucose levels, perigonadal fat weight, and liver weight), predominantly in male, whereas MCD and PUD did not induce MetS in WT. Nevertheless, LCD failed to induce MetS in KO, confirming that Cav1 KO mice are resistant to diet-induced obesity. PV LPS levels were elevated only in male WT LCD with no change in KO. There was no FITC-LPS m-to-s movement in WT SD jejunum, whereas luminal addition of OA/TCA increased LPS m-to-s movement. Interestingly, FITC-LPS m-to-s movement was increased in jejunum from WT LCD with no further increase after OA/TCA challenge, suggesting that LCD constitutively upregulated LPS transport. FD4 permeability was significantly higher in WT LCD than in WT SD, but not in KO. MCD but no other diet somewhat increased TER, compared with WT SD or KO SD. Constitutive LPS transport in WT LCD jejunum was abolished by luminal addition of the lipid raft inhibitor methyl-β-cyclodextrin (0.1 mM), suggesting the involvement of transcytotic rather than paracellular transport mechanisms. No LPS transport was observed in KO SD and KO LCD jejunum, further suggesting that LCD-induced LPS transport is Cav1 dependent. These results suggest that LCD constitutively activates Cav1-mediated endocytotic LPS transport in the small intestine, resulting in metabolic endotoxemia and the MetS. Cav1-mediated LPS transport is a promising therapeutic target for diet-induced, LPS-associated MetS. VA Merit Review and DoD grants. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exerkine FNDC5/irisin-enriched exosomes promote proliferation and inhibit ferroptosis of osteoblasts through interaction with Caveolin-1.

Postmenopausal osteoporosis is a prevalent metabolic bone disorder characterized by a decrease in bone mineral density and deterioration of bone microstructure. Despite the high prevalence of this disease, no effective treatment for osteoporosis has been developed. Exercise has long been considered a potent anabolic factor that promotes bone mass via upregulation of myokines secreted by skeletal muscle, exerting long-term osteoprotective effects and few side effects. Irisin was recently identified as a novel myokine that is significantly upregulated by exercise and could increase bone mass. However, the mechanisms underlying exercise-induced muscle-bone crosstalk remain unclear. Here, we identified that polyunsaturated fatty acids (arachidonic acid and docosahexaenoic acid) are increased in skeletal muscles following a 10-week treadmill exercise programme, which then promotes the expression and release of FNDC5/irisin. In osteoblasts, irisin binds directly to Cav1, which recruits and interacts with AMP-activated protein kinase α (AMPKα) to activate the AMPK pathway. Nrf2 is the downstream target of the AMPK pathway and increases the transcription of HMOX1 and Fpn. HMOX1 is involved in regulating the cell cycle and promotes the proliferation of osteoblasts. Moreover, upregulation of Fpn in osteoblasts enhanced iron removal, thereby suppressing ferroptosis in osteoblasts. Additionally, we confirmed that myotube-derived exosomes are involved in the transportation of irisin and enter osteoblasts through caveolae-mediated endocytosis. In conclusion, our findings highlight the crucial role of irisin, present in myotube-derived exosomes, as a crucial regulator of exercise-induced protective effects on bone, which provides novel insights into the mechanisms underlying exercise-dependent treatment of osteoporosis.

Camellia sinensis polysaccharide attenuates inflammatory responses via the ROS-mediated pathway by endocytosis

Polysaccharide CSTPs extracted from Camellia sinensis tea-leaves possessed unique against oxidative damage by scavenging ROS. Herein, acid tea polysaccharide CSTPs-2 with tightly packed molecular structure was isolated, purified and characterized in this research. Furthermore, the effects of CSTPs-2 on ROS-involved inflammatory responses and its underlying mechanisms were investigated. The results suggest that CSTPs-2 dramatically reduced the inflammatory cytokines overexpression and LPS-stimulated cell damage. CSTPs-2 could trigger the dephosphorylation of downstream AKT/MAPK/NF-κB signaling proteins and inhibit nuclear transfer of p-NF-κB to regulate the synthesis and release of inflammatory mediators in LPS-stimulated cells by ROS scavenging. Importantly, the impact of CSTPs-2 in downregulating pro-inflammatory cytokines and mitigating ROS overproduction is associated with clathrin- or caveolae-mediated endocytosis uptake mechanisms, rather than TLR-4 receptor-mediated endocytosis. This study presents a novel perspective for investigating the cellular uptake mechanism of polysaccharides in the context of anti-inflammatory mechanisms.

Tetrahedral framework nucleic acids’ role in facilitating chronic diabetic wound repair via the endoplasmic reticulum-mitochondrial pathway

Under diabetic circumstances, keratinocytes are constantly exposed to hyperglycaemic micromilieu, leading to disrupted differentiation and increased chronic wound susceptibility during to sustained endoplasmic reticulum (ER) stress (ERS) and mitochondrial dysfunction during hyperglycaemia. As mitochondrial respiratory chain activity is a primary generator of intracellular reactive oxygen species (ROS), we postulated that tFNAs could potentially boost mitochondrial function and energy metabolism in the presence of oxidative stress. To this end, we first explored the ER-mitochondrial (ER-MITO) axis-associated intracellular transportation of tetrahedral framework nucleic acids (tFNAs). tFNAs are transported to the ER and mitochondria via caveolae-mediated endocytosis, thereby regulating the ER-MITO axis. Additionally, tFNAs reduce ERS by downregulating PERK-ATF 4-CHOP, thereby stabilising mitochondrial dynamics, improving energy metabolism, maintaining membrane potential polarisation, and scavenging generated reactive oxygen species to counteract oxidative stress during hyperglycaemia. Consequently, tFNAs restore cell proliferation, migration, and differentiation and enhance chronic wound healing and skin graft survival. Furthermore, tFNAs repaired the diabetic skin mechanical barrier, underscoring their versatility of ER-MITO axis modulation. Overall, this study highlights the importance of tFNAs in ER-MITO axis modulation and a novel therapeutic approach for chronic diabetic wound healing.

Effect of Lipid Composition on the Interaction of Liposomes with THP-1-Derived Macrophages.

Recently, liposomal formulations that target macrophages have been used to treat lung diseases. However, the detailed mechanism of the cellular uptake must be elucidated to identify a formulation with excellent cellular uptake efficiency to treat non-tuberculous mycobacterial lung disease. We studied the effect of lipid composition on the cellular uptake of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (Chol) liposomes with a size of approximately 200 nm into THP-1-derived macrophages. The amount of DPPC/Chol liposomes (80/20 mol%) was greater than that of DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes. The anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane fluidity of the DPPC/Chol (80/20 mol%) liposomes was higher than that of the other two liposomes. DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes were taken up via clathrin- and caveolae-mediated endocytosis and phagocytosis. However, proteins involved in cellular uptake through ligand-receptor interactions were adsorbed to a greater extent on DPPC/Chol (80/20 mol%) liposomes than on DPPC/Chol (67/33 mol%) liposomes. Pretreatment of cells with antibodies against the low-density lipoprotein receptor and scavenger receptor type B1 largely inhibited the uptake efficiency of DPPC/Chol (80/20 mol%) liposomes. Our results indicate that the membrane fluidity of DPPC/Chol liposomes, which is controlled by the Chol ratio, is an important factor in controlling protein adsorption and the subsequent uptake efficiency of liposomes.

Tris-assisted one-step fabrication of functional carbon dots for specific folate receptor positive-expressed cancer cell imaging

Specific staining of cancer cells is momentous for cancer research. Nanoprobe with multivalent recognition is emerging as powerful tools for bioimaging, but the nonspecific cell uptake and complex functional modification procedures are still obstacles for specific detection and convenient synthesis. Carbon dots (CDs) with an intrinsic targeting ability, excellent optical properties and biocompatibility acquired from an efficient one-step fabrication procedure were urgently desired in specific cancer cells visualization. Herein, inspired by the interrelationships between interface and biomolecular mechanisms, we suggested that it was possible to construct CDs with the desired characteristics for folate receptor (FR) positive-expressed cancer cell imaging via rich hydroxyl groups Tris-assisted one-step hydrothermal treatment of folate acid (FA) and l-Arginine (L-Arg) precursors. The prepared small-sized F-CDs were equipped with abundant hydroxyl, pterin and negative charge surface, and possessed environmental friendliness, outstanding photostability and biocompatibility. Moreover, F-CDs had an intrinsic FR positive-expressed cancer cell targeting ability without any post-modification of the ligands. Rich hydroxyl groups play a vital role in endowing the optical properties and biological effects of F-CDs. F-CDs could be used as a promising candidate for FR-expressed cancer cell labeling and tracking. In addition, the caveolae-mediated endocytosis pathway of F-CDs was ascertained. More importantly, experimental results confirmed that the combination of physicochemical properties may provide an efficient strategy to overcome non-specific cell uptake interactions for cell labeling. Our strategy put forward a promising alternative to design fluorescent CDs for extensive chemical and biomedical applications.

Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages.

In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

Intracellularly delivered human lactoferrin functions as an activator of Na+/H+ exchanger 7

Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.

Caveolae-mediated endocytosis of extracellular QSOX1b modulates the migration of fibroblasts

Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.