RESUMEN
Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.
Asunto(s)
ADN/química , Lisosomas/metabolismo , Macrófagos/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Fagocitosis/fisiología , Animales , Aptámeros de Nucleótidos/farmacología , Autofagia/fisiología , Células COS , Calcio/metabolismo , Carbocianinas/farmacología , Línea Celular Tumoral , Chlorocebus aethiops , Células Hep G2 , Humanos , Lisosomas/efectos de los fármacos , Ratones , Nanocompuestos/química , Fagosomas/metabolismo , Células RAW 264.7RESUMEN
The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe Voltair, a fluorescent DNA nanodevice that reports the absolute membrane potential and can be targeted to organelles in live cells. Voltair consists of a voltage-sensitive fluorophore and a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair, we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics and enhance our understanding of how membrane potential regulates organelle biology.
Asunto(s)
ADN/química , Biología Molecular/instrumentación , Biología Molecular/métodos , Orgánulos/química , Animales , Electrofisiología/instrumentación , Electrofisiología/métodos , Endocitosis , Diseño de Equipo , Colorantes Fluorescentes , Células HEK293 , Humanos , Membranas Intracelulares/química , Lisosomas/química , Potenciales de la Membrana , Imagen de Lapso de TiempoRESUMEN
Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted) fluorochromes were identified. Their cytotoxicity was tested and found that between 1-10 micromolar range, they were non-toxic even during long-term incubations.
