In Vitro Reconstitution of Discrete Stages of Dynamin‐dependent Endocytosis
Introduction
The formation of vesicles or trafficking intermediates is a complex process, requiring interplay between numerous soluble and membrane‐bound proteins and lipids. Together these components work to generate local deformation of a membrane, capture cargo, and promote the formation of a vesicle or tubule intermediate. In cells, these processes can be examined by deleting individual components or by over‐expressing proteins or their domains. This provides information about trafficking pathways but seldom yields precise information about the individual function of a protein component. Therefore the in vitro approach is valuable, allowing more precise roles to be assigned to proteins and allowing vesicle formation to be studied in molecular detail. However, care is needed in interpreting these data as a limited subset of binding partners is used and the lipid composition of model membranes and the concentration of protein components may not reflect those present in vivo. Therefore in vivo and in vitro approaches should be used in tandem, both resulting in different information and with in vivo experiments providing crosschecks to confirm in vitro data. Despite the pitfalls of studying isolated protein components, tremendous progress has been made in cell biology by such approaches, and only by understanding the molecular details can we begin to intelligently design therapies.
Section snippets
Making Liposomes of a Defined Size
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Prepare lipid mixtures to a total lipid concentration of 1 mg/ml in a 19:1 mixture of chloroform:methanol in a glass tube. Methanol is necessary to maintain the solubility of some lipids, including PtdIns(4,5)P2. A common lipid mixture consists of 10% cholesterol, 40% PtdEth, 40% PtdSer, and 10% of the lipid specific for the protein of interest (often PtdIns(4,5)P2). An alternative is to use a Folch brain lipid extract or a liver lipid extract.
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Remove solvent from the lipid mixture by
Conclusions and Future Directions
The methods presented here provide snapshots of membrane budding and scission processes. Future developments will make the assays more quantitative rather than qualitative, allowing these studies to address many of the important questions remaining in understanding vesicle formation such as the mechanism of cargo incorporation into a coated vesicle, the energetics of vesicle generation, the use of alternative adaptor molecules, and the regulation of the process. No doubt these techniques will
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