एक जीवित कोशिकाओं के प्लाज्मा झिल्ली में फ्लोरोसेंट प्रोटीन लिपिड बेड़ा विभाजन जांच तकनीक वर्णित है. यह अंदर या के बाहर लिपिड rafts स्थित प्रोटीन का प्रसार बार में असमानता का लाभ लेता है. नियंत्रण की स्थिति में या दवा के बाद इसके अलावा अधिग्रहण गतिशील प्रदर्शन किया जा सकता है.
In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.
एफसीएस विधि यहाँ प्रस्तुत जीवित कोशिकाओं में ब्याज की जांच फ्लोरोसेंट के लिपिड बेड़ा विभाजन की एक संवेदनशील और तेजी से विश्लेषण के लिए सक्षम बनाता है. एफसीएस फोटान गिनती की संवेदनशीलता के साथ confocal माइ?…
The authors have nothing to disclose.
इस काम Agence Nationale डे ला Recherche से (ChoAD) अनुदान द्वारा समर्थित किया गया. हम भी Fondation ICM (Institut डु Cerveau एट डी ला Moelle) के उनके वित्तीय समर्थन के लिए आभारी हैं.
Name of the reagent | Company | Catalogue number | Comments |
Cholera toxin subunit B-Alexa 488 | Invitrogen | C-34775 | MW (pentamer) = 57 kg/mol |
Confocal microscope | Leica | SP5 | |
Incubator for temperature and CO2 control | Life imaging services | The Cube and the Box | |
SPAD (Single Photon Avalanche Diode) | MPD (Micro Photon Devices) | PDM serie (100 μm sensitive area) | |
High pass 488 nm filter | Semrock | 488 nm blocking edge BrightLine long-pass filter Part # FF01-488/LP-25 |
|
FCS detection unit | Picoquant | Picoharp 300 module | |
Acquisition and auto-correlation software | Picoquant | SymPhoTime | |
Fitting software | OriginLab | OriginPro8 |