Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Dominik  P. Buser; Martin Spiess

doi:10.3791/59111

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Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

8,926 Views

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11:05 min

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February 21, 2019

DOI:

10.3791/59111-v

DOI:

10.3791/59111-v

•

11:05 min

February 21, 2019

•

1 Views

Dominik P. Buser¹, Martin Spiess¹

¹Biozentrum,University of Basel

Transcript

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

Summary

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.