Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells
February 21st, 2019
Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.
This method enables the production and application of bacterial expressed, functionalised nano-bodies to analyze retrograde protein transport from the cell surface to intercellular compartments.In particular the Trans-Golgi network.The main advantage of this technique is the use of sulfation.A highly specific, compartmentalized, and sensitive post-translation modification to assess reporter nano-body complex arrival in the DTN compartment.This method in combination with other derivatised nano-bodies for life cell imaging and electron microscopy can also be used to study endocitic processes of disease-related cell surface receptors.Our repertoire of functionalised nano-bodies has been engineered to study retrograde transport in cultured cells.However, these tools may also be applied in multicellular organisms.To begin this procedure, pick a bacterial colony containing the vector of interest from the plate and inoculate a flask containing 20 milliliters of LB medium supplemented with antibiotics.Incubate overnight at 37
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Overview
This article discusses the retrograde transport of proteins from the cell surface to the Golgi apparatus, a crucial process for maintaining membrane homeostasis. A method for biochemically analyzing this transport in HeLa cells using functionalized nanobodies is described.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Biochemistry
Background
- Protein transport is vital for cellular function.
- Retrograde transport plays a key role in membrane dynamics.
- HeLa cells are a widely used model for studying cellular processes.
- Functionalized nanobodies can be used to track protein movement.
Purpose of Study
- To analyze the mechanisms of protein transport from the cell surface to the Golgi.
- To utilize functionalized nanobodies for biochemical analysis.
- To enhance understanding of membrane homeostasis in cells.
Methods Used
- Use of functionalized nanobodies to label recombinant proteins.
- Biochemical assays to track protein transport.
- Analysis of transport dynamics in HeLa cells.
- Evaluation of membrane homeostasis effects.
Main Results
- Successful labeling of proteins with functionalized nanobodies.
- Demonstrated transport of proteins to the Golgi apparatus.
- Insights into the role of retrograde transport in membrane dynamics.
- Potential applications for studying other cellular transport mechanisms.
Conclusions
- Retrograde transport is essential for maintaining cellular function.
- Functionalized nanobodies are effective tools for studying protein dynamics.
- This method can be applied to further research in cell biology.
Frequently Asked Questions
Chapters in this video
0:04
Title
1:03
Bacterial Liquid Culture and Induction of Functionalized Nanobody Expression
2:31
Purification of Functionalized Nanobodies
4:14
Validation of Functionalized Nanobody Expression
4:41
Uptake of Functionalized Nanobodies by Cultured Cells
8:03
Results: Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network
10:09
Conclusion
