Question

You have undertaken a study of protein transport in live cultured eukaryotic cells, and your objective is to follow the in vivo movement of a secreted protein through the secretory pathway to the cell exterior. You have introduced a gene that encodes a mutant form of this protein tagged with Green Fluorescent Protein (GFP) into the cells. When the cells are grown at 42^OC, the mutant protein is folded in a way that causes it to be retained in the rough ER, whereas at 32^oC, the protein folds normally and is able to proceed through the pathway. Briefly describe an experiment that would allow you to accomplish your objective and be sure to mention the microscopic technique you would use, ( I read some earlier posts I don't know why you would need to incubate at both sets of temperature) also which type of fluorescence microscopy would you use here? would it be confocal?)

Answer 1

Answer:

Experiment:

• Secretory pathway refers to the process by which proteins (lipids) are transported to the cell membrane and are released out to the extracellular environment.
• The pathway involves the movement and modification of proteins through specific membrane bound organelles and vesicles: like, from endoplasmic reticulum (ER) lumen, through transport vesicles, to cis-Golgi network, followed by trans-Golgi network.
• Sometimes protein misfolding may result in retention of the protein in the ER.
• The experiment uses the mutant protein (Vesicular Stomatitis virus G-VSVG) protein has been effectively used for the purpose.
• The VSVG protein is temperature sensitive.
• At temperature of 42°C, misfolding of the protein occurs (due to alteration of an amino acid), which allows it to be retained in the ER. While at 32°C the alteration is reversed by proper chaperon interaction, and the protein is normally transported across secretory pathway.
• The gene for the protein is tagged with GFP (Green fluorescent protein).
• These were transfected in cells and incubated at sets of temperatures of 42°C and 32°C.
• The proteins are further detected with monoclonal antibody, followed by FRET detection.
• FRET represent: Forster or fluorescence (also phosphorescence) resonance energy transfer technology.
• Biosensors based on FRET technologies, comprises of fluorophores, which can be conjugated with the molecule to be studied and observed spectroscopically by a sensing domain.
• The energy transferred by the donor to the acceptor, in measured by fluorescence emission using fluorescence microscopy, fluorescence confocal laser scanning microscopy or fluorescence lifetime imaging microscopy (FLIM).
• The normal protein (those incubated at of 32°C), interacts with chaperon and thus will not interact with antibody, retaining fluorescence of GFP.
• The mis-folded protein (those incubated at of 42°C), does not interact with chaperon ad binds to antibody, detected by antibody staining.

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