RESUMEN

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.

Asunto(s)

Células Cromafines/fisiología , Células Cromafines/ultraestructura , Endocitosis/fisiología , Células Neuroendocrinas/fisiología , Células Neuroendocrinas/ultraestructura , Animales , Señalización del Calcio , Bovinos , Fusión Celular , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Sistemas de Computación , Dinaminas/fisiología , Exocitosis/fisiología , Fusión de Membrana , Cultivo Primario de Células , Vesículas Sinápticas/metabolismo

RESUMEN

A novel bioinspired nanoplatform capable of fast endocytosis, lysosomal pH-triggered drug release, and reduced drug efflux based on PBA-PEG-b-P(Glu-co-GluDA) copolymer was developed in this study. The synthesized copolymer could facilitate doxorubicin encapsulation with relatively high drug-loading content and efficiency. Inspired by mussel byssal threads, a core crosslinking strategy based on the coordination between catechol and ferric ions was introduced to improve the stability of nanomicelles and realize lysosomal pH-controlled drug release. This nanoplatform could maintain integrity even after being dissolved in a good solvent, demonstrating its the potential to withstand infinite dilution of plasma after intravenous injection. Moreover, this nanoplatform demonstrated lysosomal pH-triggered drug release, and the cumulative release amount of doxorubicin under a simulated lysosomal condition was 13 times higher than that under a simulated plasma condition. Moreover, as a result of the high binding capacity between phenylboronic acid (PBA) and sialic acid on the surface of human hepatoma cell line (HepG2), the fast and enhanced endocytosis in addition to lysosomal pH-triggered release property and significantly low efflux, this nanoplatform exhibits improved delivery efficiency of doxorubicin into the nucleus and notably outstanding antiproliferative effects compared with doxorubicin. Furthermore, the PBA modification remarkably increased the mean fluorescence intensity of this nanoplatform endocytosed by HepG2 cells to twice that of doxorubicin after one hour of incubation. The nanoplatform exhibited an inhibition rate of 70% against tumor growth. Thus, this novel nanoplatform based on PBA-PEG-b-P(Glu-co-GluDA) copolymer displayed multifunctionality and exhibited great potential as an intelligent nanoplatform for antitumor drug delivery.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , Núcleo Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Doxorrubicina/farmacología , Lisosomas/efectos de los fármacos , Nanopartículas/química , Antibióticos Antineoplásicos/metabolismo , Materiales Biomiméticos/química , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Catecoles/química , Catecoles/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/metabolismo , Composición de Medicamentos/métodos , Endocitosis , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Hierro/química , Hierro/metabolismo , Lisosomas/metabolismo , Micelas , Nanopartículas/ultraestructura , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo

RESUMEN

Without robust mechanisms to efficiently form new synaptic vesicles (SVs), the tens to hundreds of SVs typically present at the neuronal synapse would be rapidly used up, even at modest levels of neuronal activity. SV recycling is thus critical for synaptic physiology and proper function of sensory and nervous systems. Yet, more than four decades after it was originally proposed that the SVs are formed and recycled locally at the presynaptic terminals, the mechanisms of endocytic processes at the synapse are heavily debated. Clathrin-mediated endocytosis, a type of endocytosis that capitalizes on the clathrin coat, a number of adaptor and accessory proteins, and the GTPase dynamin, is well understood, while the contributions of clathrin-independent fast endocytosis, kiss-and-run, bulk endocytosis and ultrafast endocytosis are still being evaluated. This review article revisits and summarizes the current knowledge on the SV reformation with a focus on clathrin-mediated endocytosis, and it discusses the modes of SV formation from endosome-like structures at the synapse. Given the importance of this topic, future advances in this active field are expected to contribute to better comprehension of neurotransmission, and to have general implications for neuroscience and medicine.