Visualisere Clathrin endocytose af G-protein-koblede receptorer på Single-begivenhed opløsning via TIRF Mikroskopi
Summary
Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.
Abstract
Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.
Introduction
Processen med clathrin-medieret endocytose (CME) er afhængig af den vel-timede ankomst af de mange komponenter i clathrin-medieret endocytiske maskiner at samle gods og manipulere plasmamembranen i vesiklerne 1-3. CME initieres ved membran deformere og fragt-adapter proteiner, der kommer sammen på spirende steder endocytose 1. Disse proteiner rekruttere kappeprotein clathrin, som samler i en bur-lignende struktur, der danner clathrin-coated pit (CCP) 4. Når CCP er samlet i en kugleform, membran spaltning, primært gennem indsats af det store GTPase, dynamin, genererer gratis clathrin-coatede vesikler (CCVS) 5,6. Denne internalisering udløser hurtig afmontering af clathrin frakke, så komponenter, der skal genbruges til flere runder af CME.
Opdagelsen og karakterisering af de involverede i CME-proteiner er blevet forankret i traditionel Biochemical, genetiske og mikroskopiteknikker 4-6,8. Disse analyser har belyst roller og interaktion punkter i disse endocytiske komponenter. Selvom meget nyttige til at fastlægge væsentlige dele af maskiner menneskehandel, er disse analyser stærkt begrænset i at opfange dynamiske opførsel CME komponenter eller koncentration last. Dette er et kritisk begrænsning, idet CME drives af koreograferede samling af sæt af protein moduler i definerede trin, og da små ændringer i dynamikken i enkelte endocytiske begivenheder kan have store kumulative konsekvenser for endocytose. Endvidere seneste data viser, at enkelte CCP'er kan variere både i sammensætning og i adfærd, hvilket tyder på, at den fysiologiske regulering af denne proces er yderst rumligt og tidsligt begrænset 9-14. Visualisere individuelle endocytiske begivenheder, derfor er vigtigt at forstå, hvorfor der er flere overflødige proteiner involveret i CME, og hvordan disse proteiner kan styres By fysiologiske signaler at regulere last internalisering.
Her beskriver vi brugen af total intern refleksion Fluorescens mikroskopi (TIRFM) for at observere CME på niveauet af dynamikken i de enkelte CCP'er i levende celler. TIRFM afhængig af forskellen i brydningsindeks mellem dækglas og det flydende celler 15,16. Når excitationslys er rettet mod cellerne ved mere end den kritiske vinkel, er det internt reflekteret, hvilket skaber en kortvarig bølge, der opretholder et tyndt område af belysning strækker ca. 100 nm over dækglasset. Dette sikrer, at kun de fluorescerende molekyler inden for dette snævre område er spændt. I praksis tillader dette excitation af fluorescerende molekyler på eller i nærheden af plasmamembranen, og minimerer fluorescens fra de indre dele af cellen. Dette giver en signifikant højere signal-støj-forholdet og z-aksen beslutning at visualisere events på plasmamembranen, compared til mere almindeligt anvendte transportformer såsom konventionel epifluorescens eller konfokal fluorescens mikroskopi. Vi beskriver også, ved en indledende og praktisk niveau, at anvendelsen af et almindeligt anvendt billedanalyse platform analysere og kvantificere simple morfologiske træk og dynamikken i de enkelte last endocytiske begivenheder.
Protocol
Representative Results
Discussion
Her beskriver vi brugen af TIRFM at visualisere clathrin endocytose (CME) på niveau med enkelte CCP'er i levende celler i realtid. CME er en hurtig og meget dynamisk begivenhed medieret af den kumulative virkning af mange rumligt og tidsligt adskilte enkelte arrangementer. De fleste analyser, der i øjeblikket bruges, såsom biokemiske målinger af internalisering ved hjælp af overflade biotinylering eller ligand binding, flow-cytometri eller fikserede celler assays måler mængden af internaliserede p…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Drs. C. Szalinski, H. Teng, and M. Bruchez for help with Imaris, R. Vistein, and D. Shiwarski for technical help and advice, and Dr. M von Zastrow, Dr. T Kirchhausen, Dr. D Drubin, and Dr. W Almers for reagents and helpful discussion. Funding provided by T32 grant NS007433 to SLB and NIH DA024698 and DA036086 to MAP.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| DMEM/High Glucose with L-glutamine and sodium pyruvate | Fisher Scientific | SH3024301 | |
| Dulbecco's Phosphate Buffered Saline (DPBS), no calcium, no magnesium | Gibco, by Life Technologies | 21600-010 | |
| EDTA Free Acid | Amresco | 0322-500G | |
| Fetal Bovine Serum | Gibco, by Life Technologies | 10437-028 | |
| Leibovitz's L-15 Medium, no phenol red | Gibco, by Life Technologies | 21083-027 | |
| Opti-MEM | Gibco, by Life Technologies | ||
| HEPES | CellPURE by Fisher Scientific | BP2937-100 | |
| Effectene | Qiagen | 301425 | Transfection reagent |
| 25 millimeter coverglass | Fisher Scientific | 12-545-86 | |
| Corning cell culture treated flasks, 25cm2 | Fisher Scientific | 10-126-28 | |
| Cell culture 6-well plate | Greiner Bio-One, by VWR | 82050-896 | |
| Monoclonal ANTI-FLAG M1 | Sigma Aldrich | F3040-5MG | |
| [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) | Sigma Aldrich | E7384-5MG | |
| Alexa Fluor 647 Protein Labeling Kit | Life Technologies | A20173 | |
| ImageJ | NIH | http://rsb.info.nih.gov/ij/ | |
| Imaris Image analysis software | BitPLane | http://www.bitplane.com/imaris/imaris, for automated analysis | |
| Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters | Nikon | ||
| Nikon TIRF arm with required adapters for Nikon Eclipse Ti | Nikon | For adjusting angle of incidence | |
| iXon+ EMCCD camera and adapters | Andor |
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