RESUMO
Purpose: Castration Resistant Prostate Cancer (CRPC) is characterized by poor prognosis and limited therapeutic options. AgNPs functionalized with glucose (G-AgNPs) were observed cytotoxic to CRPC cell lines (PC-3 and Du-145) and not LNCaP. This study aims to evaluate AgNPs and G-AgNPs' uptake mechanisms in these cells and understand their role in the selective effect against CRPC cells. Methods: Uptake of AgNPs and G-AgNPs was assessed through transmission electron microscopy (TEM). A microRNA (miRNAs) analysis approach was used to uncover the main molecular differences responsible for the endocytic mechanisms' regulation. Caveolin (Cav) 1 and 2 mRNA and protein levels were assessed in the three cell lines. Caveolae-dependent endocytosis was inhibited with genistein or siCav1- and siCav2- in PC-3 and Du-145 and resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration. Caveolae-dependent endocytosis was induced with Cav1+ and Cav2+ plasmids in LNCaP, resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration and TEM to assess their location. Results: AgNPs and G-AgNPs were not uptaked by LNCaP. miRNA analysis revealed 37 upregulated and 90 downregulated miRNAs. Functional enrichment analysis of miRNAs' targets resulted in enrichment of terms related to endocytosis and caveolae. We observed that Cav1 and Cav2 are not expressed in LNCaP. Inhibiting caveolae-dependent endocytosis in Du-145 and PC-3 led to a significative reduction of cytotoxic capacity of AgNPs and G-AgNPs and induction of caveolae-dependent endocytosis in LNCaP lead to a significative increase as well as their uptake by cells. Conclusion: This study shows the potential of these AgNPs as a new therapeutic approach directed to CRPC patients, uncovers caveolae-dependent endocytosis as the uptake mechanism of these AgNPs and highlights deregulation of Cav1 and Cav2 expression as a key difference in hormone sensitive and resistant PCa cells which may be responsible for drug resistance.
Assuntos
Cavéolas , Caveolina 1 , Endocitose , Nanopartículas Metálicas , MicroRNAs , Neoplasias de Próstata Resistentes à Castração , Prata , Masculino , Humanos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Cavéolas/metabolismo , Cavéolas/efeitos dos fármacos , Prata/química , Prata/farmacologia , Prata/farmacocinética , Caveolina 1/metabolismo , Caveolina 1/genética , Nanopartículas Metálicas/química , Linhagem Celular Tumoral , MicroRNAs/metabolismo , MicroRNAs/genética , Sobrevivência Celular/efeitos dos fármacos , Caveolina 2/metabolismo , Caveolina 2/genética , Antineoplásicos/farmacologia , Células PC-3RESUMO
In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.
Assuntos
Esclerose Lateral Amiotrófica , Modelos Animais de Doenças , Endocitose , Neurônios Motores , Vesículas Sinápticas , Animais , Camundongos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Diafragma/inervação , Diafragma/metabolismo , Diafragma/fisiopatologia , Endocitose/fisiologia , Corantes Fluorescentes/metabolismo , Imidazóis/farmacologia , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Terminações Nervosas/metabolismo , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Transmissão Sináptica/fisiologia , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismoRESUMO
Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. End4p directly transmits force in CME by binding to both the membrane (through the AP180 N-terminal homology [ANTH] domain) and F-actin (through the talin-HIP1/R/Sla2p actin-tethering C-terminal homology [THATCH] domain). We show that 7 pN force is required for stable binding between THATCH and F-actin. We also characterized a domain in End4p, Rend (rod domain in End4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15 pN terminates the transmission of force. We show that the mechanical properties (mechanical stability, unfolding extension, hysteresis) of Rend and THATCH are tuned to form a circuit for the initiation, transmission, and termination of force between the actin cytoskeleton and membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.
Assuntos
Actinas , Clatrina , Endocitose , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Endocitose/fisiologia , Schizosaccharomyces/metabolismo , Clatrina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Actinas/metabolismo , Domínios Proteicos , Citoesqueleto de Actina/metabolismo , Ligação Proteica , Membrana Celular/metabolismoRESUMO
Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion.
Assuntos
Proteínas de Drosophila , Endossomos , Transporte Proteico , Proteína Wnt1 , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Asas de Animais/metabolismo , Proteína Wnt1/metabolismo , Proteína Wnt1/genéticaRESUMO
Mitochondria, the dynamic organelles responsible for energy production and cellular metabolism, have the metabolic function of extracting energy from nutrients and synthesizing crucial metabolites. Nevertheless, recent research unveils that intercellular mitochondrial transfer by tunneling nanotubes, tumor microtubes, gap junction intercellular communication, extracellular vesicles, endocytosis and cell fusion may regulate mitochondrial function within recipient cells, potentially contributing to disease treatment, such as nonalcoholic steatohepatitis, glioblastoma, ischemic stroke, bladder cancer and neurodegenerative diseases. This review introduces the principal approaches to intercellular mitochondrial transfer and examines its role in various diseases. Furthermore, we provide a comprehensive overview of the inhibitors and activators of intercellular mitochondrial transfer, offering a unique perspective to illustrate the relationship between intercellular mitochondrial transfer and diseases.
Assuntos
Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Animais , Comunicação Celular , Vesículas Extracelulares/metabolismo , Transporte Biológico , Endocitose/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapiaRESUMO
Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.
Assuntos
Células Cromafins , Dinamina II , Endocitose , Exocitose , Mutação , Miopatias Congênitas Estruturais , Células Cromafins/metabolismo , Endocitose/fisiologia , Endocitose/genética , Dinamina II/genética , Dinamina II/metabolismo , Animais , Exocitose/fisiologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/metabolismo , Mutação/genética , Bovinos , Humanos , Actinas/metabolismo , Actinas/genética , Células Cultivadas , Técnicas de Patch-Clamp , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologiaRESUMO
Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.
Assuntos
Angiotensina II , Endocitose , Proteínas de Membrana , Receptor Tipo 1 de Angiotensina , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Humanos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Camundongos , Albuminúria/metabolismo , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Compostos de Bifenilo/farmacologia , Irbesartana/farmacologia , Células HEK293 , beta-Arrestina 2/metabolismo , beta-Arrestina 2/genética , Benzimidazóis , TetrazóisRESUMO
ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.
Assuntos
Canais de Cloreto , Endocitose , Xenopus laevis , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Endocitose/fisiologia , Camundongos , Túbulos Renais Distais/metabolismo , Hiperplasia , Humanos , Feminino , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Camundongos Endogâmicos C57BL , Células HEK293 , Oócitos/metabolismo , Proteínas de Transporte de ÂnionsRESUMO
Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and ß2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.
Assuntos
Complexo 2 de Proteínas Adaptadoras , Chaperonas Moleculares , Complexo 2 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Ligação Proteica , Endocitose/fisiologia , Transporte ProteicoRESUMO
Ribbon synapses of inner hair cells (IHCs) are uniquely designed for ultrafast and indefatigable neurotransmission of the sound. The molecular machinery ensuring the efficient, compensatory recycling of the synaptic vesicles (SVs), however, remains elusive. This study showed that hair cell knock-out of murine Dmxl2, whose human homolog is responsible for nonsyndromic sensorineural hearing loss DFNA71, resulted in auditory synaptopathy by impairing synaptic endocytosis and recycling. The mutant mice in the C57BL/6J background of either sex had mild hearing loss with severely diminished wave I amplitude of the auditory brainstem response. Membrane capacitance measurements of the IHCs revealed deficiency in sustained synaptic exocytosis and endocytic membrane retrieval. Consistent with the electrophysiological findings, 3D electron microscopy reconstruction showed reduced reserve pool of SVs and endocytic compartments, while the membrane-proximal and ribbon-associated vesicles remain intact. Our results propose an important role of DMXL2 in hair cell endocytosis and recycling of the SVs.
Assuntos
Endocitose , Células Ciliadas Auditivas Internas , Proteínas do Tecido Nervoso , Vesículas Sinápticas , Animais , Feminino , Masculino , Camundongos , Endocitose/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Exocitose/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vesículas Sinápticas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Dysregulation of the hematopoietic niche during hyperlipidemia facilitates pathologic leukocyte production, driving atherogenesis. Although definitive hematopoiesis occurs primarily in the bone marrow, during atherosclerosis this also occurs in the spleen. Cells of the bone marrow niche, particularly endothelial cells, have been studied in atherosclerosis, although little is known about how splenic endothelial cells respond to the atherogenic environment. Here we show unique dysregulated pathways in splenic compared to bone marrow endothelial cells during atherosclerosis, including perturbations of lipid metabolism and endocytic trafficking pathways. As part of this response, we identify the mixed lineage kinase domain-like (MLKL) protein as a repressor of splenic, but not bone marrow, myelopoiesis. Silencing MLKL in splenic endothelial cells results in inefficient endosomal trafficking and lipid accumulation, ultimately promoting the production of myeloid cells that participate in plaque development. These studies identify endocytic trafficking by MLKL as a key mechanism of splenic endothelial cell maintenance, splenic hematopoiesis and, subsequently, atherosclerosis.
Assuntos
Aterosclerose , Células Endoteliais , Hiperlipidemias , Proteínas Quinases , Baço , Baço/patologia , Baço/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Animais , Aterosclerose/patologia , Aterosclerose/metabolismo , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Masculino , Mielopoese , Humanos , Células Cultivadas , Metabolismo dos Lipídeos , Camundongos , Placa Aterosclerótica/patologia , Placa Aterosclerótica/metabolismo , Camundongos Knockout para ApoE , Endocitose/fisiologia , Endossomos/metabolismo , Nicho de Células-Tronco/fisiologiaRESUMO
AIMS: Hypoperfusion induces significant white matter injury in cerebral vascular disorders, including arteriosclerotic cerebral small vessel disease (aCSVD), which is prevalent among the elderly. Iron transport by blood vessel endothelial cells (BVECs) from the periphery supports oligodendrocyte maturation and white matter repair. This study aims to elucidate the association between iron homeostasis changes and white matter injury severity, and explore the crosstalk between BVECs and oligodendroglial lineage cells. METHODS: In vivo: C57BL/6 mice were subjected to unilateral common carotid artery occlusion (UCCAO). In vitro: BVECs with myelin pretreatment were co-cultured with oligodendrocyte progenitor cells (OPCs) or organotypic cerebellar slices subjected to oxygen and glucose deprivation. RESULTS: Circulatory iron tends to be stored in aCSVD patients with white matter injury. Myelin debris endocytosis by BVECs impairs iron transport, trapping iron in the blood and away from the brain, worsening oligodendrocyte iron deficiency in hypoperfusion-induced white matter injury. Iron accumulation in BVECs triggers ferroptosis, suppressing iron transport and hindering white matter regeneration. Intranasal holo-transferrin (hTF) administration bypassing the BBB alleviates oligodendrocyte iron deficiency and promotes myelin regeneration in hypoperfusion-induced white matter injury. CONCLUSION: The iron imbalance between BVECs and oligodendroglial lineage cells is a potential therapeutic target in hypoperfusion-induced white matter injury.
Assuntos
Endocitose , Células Endoteliais , Ferro , Camundongos Endogâmicos C57BL , Bainha de Mielina , Oligodendroglia , Substância Branca , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Camundongos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Substância Branca/metabolismo , Substância Branca/patologia , Ferro/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Endocitose/fisiologia , Endocitose/efeitos dos fármacos , Masculino , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Células Precursoras de Oligodendrócitos/metabolismo , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/patologiaRESUMO
Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs.
Assuntos
Endocitose , Hipocampo , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Vesículas Sinápticas , Sinaptotagmina I , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Endocitose/fisiologia , Animais , Ratos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Hipocampo/metabolismo , Neurônios/metabolismo , Membrana Celular/metabolismo , Células CultivadasRESUMO
The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause the denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for the maintenance of PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes the mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p, the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9Δ cells, suggesting that defective PM integrity in vps9Δ cells is caused by lack of free ubiquitin.
Assuntos
Membrana Celular , Endocitose , Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas rab5 de Ligação ao GTP , Endocitose/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Membrana Celular/metabolismo , Endossomos/metabolismo , Resposta ao Choque Térmico/fisiologia , Vacúolos/metabolismo , Vacúolos/genética , Temperatura Alta , Ubiquitina/metabolismo , Proteínas rab de Ligação ao GTPRESUMO
Receptor-mediated endocytosis provides a mechanism for the selective uptake of specific molecules thereby controlling the composition of the extracellular environment and biological processes. The low-density lipoprotein receptor-related protein 1 (LRP1) is a widely expressed endocytic receptor that regulates cellular events by modulating the levels of numerous extracellular molecules via rapid endocytic removal. LRP1 also participates in signalling pathways through this modulation as well as in the interaction with membrane receptors and cytoplasmic adaptor proteins. LRP1 SNPs are associated with several diseases and conditions such as migraines, aortic aneurysms, cardiopulmonary dysfunction, corneal clouding, and bone dysmorphology and mineral density. Studies using Lrp1 KO mice revealed a critical, nonredundant and tissue-specific role of LRP1 in regulating various physiological events. However, exactly how LRP1 functions to regulate so many distinct and specific processes is still not fully clear. Our recent proteomics studies have identified more than 300 secreted proteins that either directly interact with LRP1 or are modulated by LRP1 in various tissues. This review will highlight the remarkable ability of this receptor to regulate secreted molecules in a tissue-specific manner and discuss potential mechanisms underpinning such specificity. Uncovering the depth of these "hidden" specific interactions modulated by LRP1 will provide novel insights into a dynamic and complex extracellular environment that is involved in diverse biological and pathological processes.
Assuntos
Endocitose , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Animais , Humanos , Endocitose/fisiologia , Camundongos , Transdução de Sinais , Especificidade de Órgãos , Camundongos KnockoutRESUMO
Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission. Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells. However, visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling. In this protocol, we describe the technical details of stimulated emission depletion (STED) imaging of synaptic endocytosis at the single-vesicle level, from sample preparation and microscopy calibration to data acquisition and analysis.
Assuntos
Endocitose , Sinapses , Vesículas Sinápticas , Endocitose/fisiologia , Animais , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Exocitose/fisiologia , Neurônios/metabolismo , Microscopia de Fluorescência/métodosRESUMO
Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked ß-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Engenharia de Proteínas , Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Células HEK293 , Engenharia de Proteínas/métodos , Endocitose/fisiologia , Transporte Proteico , AnimaisRESUMO
Synthesizing the recent progresses, we present our perspectives on how local modulations of membrane curvature, tension, and bending energy define the feedback controls over membrane traffic processes. We speculate the potential mechanisms of, and the control logic behind, the different membrane mechanics-mediated feedback in endocytosis and exo-endocytosis coupling. We elaborate the path forward with the open questions for theoretical considerations and the grand challenges for experimental validations.