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1.
Nat Commun ; 12(1): 6065, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663803

RESUMO

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Actomiosina/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Citocinese , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Fosfatidilinositóis , Monoéster Fosfórico Hidrolases
2.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638858

RESUMO

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson's disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.


Assuntos
Membrana Celular/metabolismo , Hidrazonas/farmacologia , Lisossomos/metabolismo , Naftóis/farmacologia , Canais de Potássio/metabolismo , 4-Aminopiridina/farmacologia , Animais , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Microscopia Confocal/métodos , Oócitos/citologia , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Transporte Proteico/efeitos dos fármacos , Xenopus laevis
3.
J Neurochem ; 159(1): 78-89, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34468992

RESUMO

Synaptobrevin-2 (Syb2) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is essential for neurotransmitter release. It is the most numerous protein on a synaptic vesicle (SV) and drives SV fusion via interactions with its cognate SNARE partners on the presynaptic plasma membrane. Synaptophysin (Syp) is the second most abundant protein on SVs; however, in contrast to Syb2, it has no obligatory role in neurotransmission. Syp interacts with Syb2 on SVs, and the molecular nature of its interaction with Syb2 and its physiological role has been debated for decades. However, recent studies have revealed that the sole physiological role of Syp at the presynapse is to ensure the efficient retrieval of Syb2 during SV endocytosis. In this review, current theories surrounding the role of Syp in Syb2 trafficking will be discussed, in addition to the debate regarding the molecular nature of their interaction. A unifying model is presented that describes how Syp controls Syb2 function as part of an integrated mechanism involving key molecular players such as intersectin-1 and AP180/CALM. Finally, key future questions surrounding the role of Syp-dependent Syb2 trafficking will be posed, with respect to brain function in health and disease.


Assuntos
Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/fisiologia , Proteínas SNARE/metabolismo , Sinaptofisina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Endocitose/fisiologia , Humanos , Proteínas SNARE/genética , Sinapses/genética , Sinapses/metabolismo , Sinaptofisina/genética , Proteína 2 Associada à Membrana da Vesícula/genética
4.
Dev Cell ; 56(17): 2419-2426.e4, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34473942

RESUMO

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300-880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes.


Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Saccharomyces cerevisiae/metabolismo
6.
Neuron ; 109(19): 3119-3134.e5, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34411513

RESUMO

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.


Assuntos
Células Cromafins/fisiologia , Células Cromafins/ultraestrutura , Endocitose/fisiologia , Células Neuroendócrinas/fisiologia , Células Neuroendócrinas/ultraestrutura , Animais , Sinalização do Cálcio , Bovinos , Fusão Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Sistemas Computacionais , Dinaminas/fisiologia , Exocitose/fisiologia , Fusão de Membrana , Cultura Primária de Células , Vesículas Sinápticas/metabolismo
7.
Cells ; 10(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34359976

RESUMO

Two-pore channels (TPCs) constitute a small family of ion channels within membranes of intracellular acidic compartments, such as endosomes and lysosomes. They were shown to provide transient and locally restricted Ca2+-currents, likely responsible for fusion and/or fission events of endolysosomal membranes and thereby for intracellular vesicle trafficking. Genetic deletion of TPCs not only affects endocytosis, recycling, and degradation of various surface receptors but also uptake and impact of bacterial protein toxins and entry and intracellular processing of some types of viruses. This review points to important examples of these trafficking defects on one part but mainly focuses on the resulting impact of the TPC inactivation on receptor expression and receptor signaling. Thus, a detailed RNA sequencing analysis using TPC1-deficient fibroblasts uncovered a multitude of changes in the expression levels of surface receptors and their pathway-related signaling proteins. We refer to several classes of receptors such as EGF, TGF, and insulin as well as proteins involved in endocytosis.


Assuntos
Canais de Cálcio/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Humanos , NADP/metabolismo
8.
Biomed Pharmacother ; 139: 111667, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243608

RESUMO

Interactions between vascular smooth muscle cells (VSMCs), endothelial cells (ECs), pericytes (PCs) and macrophages (MФ), the major components of blood vessels, play a crucial role in maintaining vascular structural and functional homeostasis. Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1), a transmembrane receptor protein belonging to the LDL receptor family, plays multifunctional roles in maintaining endocytosis, homeostasis, and signal transduction. Accumulating evidence suggests that LRP1 modulates vascular homeostasis mainly by regulating vasoactive substances and specific intracellular signaling pathways, including the plasminogen activator inhibitor 1 (PAI-1) signaling pathway, platelet-derived growth factor (PDGF) signaling pathway, transforming growth factor-ß (TGF-ß) signaling pathway and vascular endothelial growth factor (VEGF) signaling pathway. The aim of the present review is to focus on recent advances in the discovery and mechanism of vascular homeostasis regulated by LRP1-dependent signaling pathways. These recent discoveries expand our understanding of the mechanisms controlling LRP1 as a target for studies on vascular complications.


Assuntos
Homeostase/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Músculo Liso Vascular/metabolismo , Transdução de Sinais/fisiologia , Animais , Endocitose/fisiologia , Células Endoteliais/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Miócitos de Músculo Liso/metabolismo
9.
Nat Commun ; 12(1): 4327, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267202

RESUMO

Trivalent rare earth elements (REEs) are widely used in agriculture. Aerially applied REEs enter leaf epidermal cells by endocytosis and act systemically to improve the growth of the whole plant. The mechanistic basis of their systemic activity is unclear. Here, we show that treatment of Arabidopsis leaves with trivalent lanthanum [La(III)], a representative of REEs, triggers systemic endocytosis from leaves to roots. La(III)-induced systemic endocytosis requires AtrbohD-mediated reactive oxygen species production and jasmonic acid. Systemic endocytosis impacts the accumulation of mineral elements and the development of roots consistent with the growth promoting effects induced by aerially applied REEs. These findings provide insights into the mechanistic basis of REE activity in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Lantânio/farmacologia , NADPH Oxidases/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Endocitose/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Minerais/metabolismo , NADPH Oxidases/genética , Oxilipinas/metabolismo , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais
10.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298880

RESUMO

BACKGROUND: The proximal tubule (PT) is the major target of cadmium (Cd2+) nephrotoxicity. Current dogma postulates that Cd2+ complexed to metallothionein (MT) (CdMT) is taken up through receptor-mediated endocytosis (RME) via the PT receptor megalin:cubilin, which is the predominant pathway for reuptake of filtered proteins in the kidney. Nevertheless, there is evidence that the distal parts of the nephron are also sensitive to damage induced by Cd2+. In rodent kidneys, another receptor for protein endocytosis, the 24p3 receptor (24p3R), is exclusively expressed in the apical membranes of distal tubules (DT) and collecting ducts (CD). Cell culture studies have demonstrated that RME and toxicity of CdMT and other (metal ion)-protein complexes in DT and CD cells is mediated by 24p3R. In this study, we evaluated the uptake of labeled CdMT complex through 24p3R after acute kidney injury (AKI) induced by gentamicin (GM) administration that disrupts PT function. Subcutaneous administration of GM at 10 mg/kg/day for seven days did not alter the structural and functional integrity of the kidney's filtration barrier. However, because of PT injury, the concentration of the renal biomarker Kim-1 increased. When CdMT complex coupled to FITC was administered intravenously, both uptake of the CdMT complex and 24p3R expression in DT increased and also colocalized after PT injury induced by GM. Although megalin decreased in PT after GM administration, urinary protein excretion was not changed, which suggests that the increased levels of 24p3R in the distal nephron could be acting as a compensatory mechanism for protein uptake. Altogether, these results suggest that PT damage increases the uptake of the CdMT complex through 24p3R in DT (and possibly CD) and compensate for protein losses associated with AKI.


Assuntos
Cádmio/metabolismo , Endocitose/fisiologia , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Metalotioneína/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Néfrons/metabolismo
11.
Plant Sci ; 310: 110982, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315598

RESUMO

The spotted leaf lesion mimic trait simulates cell death in a plant responding to pathogen infection. Some spotted leaf genes are recessive, while others are dominant. A small number of plants with a lesion mimic phenotype appeared in a segregating population obtained by crossing two normal green wheat strains, XN509 and N07216. Here, we clarified the genetic model and its breeding value. Phenotyping of the consecutive progeny populations over six cropping seasons showed that the spotted leaf lesion mimic phenotype was controlled by a dominant gene designated TaSpl1, which was inhibited by two other dominant genes, designated TaSpl1-I1 and TaSpl1-I2. Using bulked segregant analysis RNA-seq (BSR-Seq) and newly developed KASP-PCR markers, the TaSpl1 and TaSpl1-I1 loci in N07216 were mapped to the end of chromosomes 3DS and 3BS, respectively. Plants with the spotted phenotype showed lower levels of stripe rust and powdery mildew than those with the normal green phenotype. Compared with normal leaves, the differentially expressed genes in spotted leaves were significantly enriched in plant-pathogen interaction and endocytosis pathways. There were no differences in the yield parameters of the F5 and F6 sister lines, N13039S with TaSpl1 and N13039 N without TaSpl1. These results provide a greater understanding of spotted leaf phenotyping and the breeding value of the lesion mimic allele in developing disease-resistance varieties.


Assuntos
Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Triticum/metabolismo , Triticum/microbiologia , Morte Celular/genética , Morte Celular/fisiologia , Resistência à Doença/fisiologia , Endocitose/fisiologia
12.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34269385

RESUMO

Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.


Assuntos
Clatrina/metabolismo , Desenvolvimento Embrionário/fisiologia , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Mamíferos , Endossomos/metabolismo , Feminino , Fertilização , Proteínas da Membrana Plasmática de Transporte de Glicina , Masculino , Camundongos , Oócitos , Proteína Quinase C , Ubiquitina/metabolismo , Ubiquitinação
13.
Nat Cell Biol ; 23(8): 859-869, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34253896

RESUMO

Dynamin has an important role in clathrin-mediated endocytosis by cutting the neck of nascent vesicles from the cell membrane. Here, using gold nanorods as cargos to image dynamin action during live clathrin-mediated endocytosis, we show that, near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle increases substantially, accompanied by simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by the coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.


Assuntos
Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Dinaminas/fisiologia , Endocitose/fisiologia , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Humanos , Microscopia/métodos , Nanotubos , Torque
14.
PLoS Pathog ; 17(7): e1009706, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252168

RESUMO

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Internalização do Vírus/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/fisiologia , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Cloroquina/farmacologia , Clatrina/metabolismo , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidroxicloroquina/administração & dosagem , Macrolídeos/farmacologia , Niclosamida/administração & dosagem , Niclosamida/farmacologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Células Vero
15.
Carbohydr Polym ; 269: 118268, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34294300

RESUMO

Major obstacles in the development of nanoformulations as efficient drug delivery systems are the rapid clearance from blood circulation and lysosomal entrapment. To overcome these problems, a polysaccharide-based core-shell type charge-switchable nanoformulation (CS-LA-DMMA/CMCS/PAMAM@DOX) is constructed to improve antitumor efficacy of DOX. By applying carboxymethyl chitosan (CMCS) as bridge polymer and negatively charged chitosan-derivative as outer shell, the stability and pH-sensitivity of this nanoformulation is promisingly enhanced. Furthermore, the positively charged PAMAM@DOX could escape from lysosomes via "proton sponge effect" and "cationic-anionic interaction with lysosome membranes". Admirable cellular uptake and high apoptosis/necrosis rate were detected in this study. In vitro assays demonstrate that the CS-LA-DMMA/CMCS/PAMAM@DOX was internalized into HepG2 cells predominantly via the clathrin-mediated endocytosis pathway. Excitingly, in vivo studies showed that high accumulation of CS-LA-DMMA/CMCS/PAMAM@DOX in tumor tissue led to enhanced tumor inhibition. Compared with free DOX, the tumor inhibition rate of nanoformulation was improved up to 226%.


Assuntos
Antineoplásicos/uso terapêutico , Quitosana/análogos & derivados , Doxorrubicina/uso terapêutico , Portadores de Fármacos/química , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sequência de Carboidratos , Quitosana/síntese química , Quitosana/química , Quitosana/metabolismo , Dendrímeros/síntese química , Dendrímeros/química , Dendrímeros/metabolismo , Doxorrubicina/química , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Endocitose/fisiologia , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Masculino , Anidridos Maleicos/química , Anidridos Maleicos/metabolismo , Camundongos Endogâmicos BALB C , Necrose/induzido quimicamente , Neoplasias/diagnóstico por imagem , Poliaminas/síntese química , Poliaminas/química , Poliaminas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Elife ; 102021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34324418

RESUMO

Synaptic membrane-remodeling events such as endocytosis require force-generating actin assembly. The endocytic machinery that regulates these actin and membrane dynamics localizes at high concentrations to large areas of the presynaptic membrane, but actin assembly and productive endocytosis are far more restricted in space and time. Here we describe a mechanism whereby autoinhibition clamps the presynaptic endocytic machinery to limit actin assembly to discrete functional events. We found that collective interactions between the Drosophila endocytic proteins Nwk/FCHSD2, Dap160/intersectin, and WASp relieve Nwk autoinhibition and promote robust membrane-coupled actin assembly in vitro. Using automated particle tracking to quantify synaptic actin dynamics in vivo, we discovered that Nwk-Dap160 interactions constrain spurious assembly of WASp-dependent actin structures. These interactions also promote synaptic endocytosis, suggesting that autoinhibition both clamps and primes the synaptic endocytic machinery, thereby constraining actin assembly to drive productive membrane remodeling in response to physiological cues.


Assuntos
Actinas/genética , Actinas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Endocitose/genética , Sinapses/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endocitose/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/metabolismo
17.
J Virol ; 95(20): e0083721, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34319159

RESUMO

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.


Assuntos
Viroses/metabolismo , Replicação Viral/fisiologia , Células A549 , Antígenos de Diferenciação/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Vírus da Parainfluenza 3 Humana/metabolismo , Vírus da Parainfluenza 3 Humana/patogenicidade , Proteínas de Ligação a RNA/metabolismo , Internalização do Vírus
18.
Arch Biochem Biophys ; 709: 108966, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34139199

RESUMO

Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steady-state levels. The protein machineries responsible for exo- and endocytosis have been extensively investigated. In contrast, less is known about the role of lipids in synaptic transmission and how the lipid composition of SVs is affected by dynamic exo-endocytotic cycling. Here we summarize the current knowledge about the composition, organization, and function of SV membrane lipids. We also cover lipid biogenesis and maintenance during the synaptic vesicle cycle.


Assuntos
Membranas Sinápticas/química , Vesículas Sinápticas/química , Animais , Endocitose/fisiologia , Exocitose/fisiologia , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo
19.
Nat Rev Mol Cell Biol ; 22(9): 625-643, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34075221

RESUMO

Endocytosis allows cells to transport particles and molecules across the plasma membrane. In addition, it is involved in the termination of signalling through receptor downmodulation and degradation. This traditional outlook has been substantially modified in recent years by discoveries that endocytosis and subsequent trafficking routes have a profound impact on the positive regulation and propagation of signals, being key for the spatiotemporal regulation of signal transmission in cells. Accordingly, endocytosis and membrane trafficking regulate virtually every aspect of cell physiology and are frequently subverted in pathological conditions. Two key aspects of endocytic control over signalling are coming into focus: context-dependency and long-range effects. First, endocytic-regulated outputs are not stereotyped but heavily dependent on the cell-specific regulation of endocytic networks. Second, endocytic regulation has an impact not only on individual cells but also on the behaviour of cellular collectives. Herein, we will discuss recent advancements in these areas, highlighting how endocytic trafficking impacts complex cell properties, including cell polarity and collective cell migration, and the relevance of these mechanisms to disease, in particular cancer.


Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Animais , Transporte Biológico , Movimento Celular , Polaridade Celular , Humanos , Morfogênese , Transdução de Sinais
20.
ACS Appl Mater Interfaces ; 13(24): 28802-28817, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34109788

RESUMO

In this study, a novel class of multifunctional responsive nanoparticles is designed and fabricated as drug nanocarriers for synergetic chemo-photothermal therapy of tumors. The proposed nanoparticles are composed of a thermo-/pH-responsive poly(N-isopropylacrylamide-co-acrylic acid) (PNA) nanogel core, a polydopamine (PDA) layer for photothermal conversion, and an outer folic acid (FA) layer as a targeting agent for the folate receptors on tumor cells. The fabricated nanoparticles show good biocompatibility and outstanding photothermal conversion efficiency. The proposed nanoparticles loaded with doxorubicin (DOX) drug molecules are stable under physiological conditions with low leakage of drugs, while rapidly release drugs in environments with low pH conditions and at high temperature. The experimental results show that the drug release process is mainly governed by Fickian diffusion. In vitro cell experimental results demonstrate that the PNA-DOX@PDA-FA nanoparticles can be phagocytized by 4T1 tumor cells and release drugs in tumor cell acidic environments, and confirm that the combined chemo and photothermal therapeutic efficacy of PNA-DOX@PDA-FA nanoparticles is higher than the photothermal therapeutic efficacy or the chemotherapeutic efficacy alone. The proposed multifunctional responsive nanoparticles in this study provide a novel class of drug nanocarriers as a promising tool for synergetic chemo-photothermal therapy of tumors.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Nanopartículas Multifuncionais/química , Acrilamidas/química , Acrilamidas/metabolismo , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/efeitos da radiação , Liberação Controlada de Fármacos , Endocitose/fisiologia , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Indóis/química , Indóis/metabolismo , Indóis/efeitos da radiação , Raios Infravermelhos , Camundongos , Nanopartículas Multifuncionais/metabolismo , Terapia Fototérmica , Polímeros/química , Polímeros/metabolismo , Polímeros/efeitos da radiação , Temperatura
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