Analysis of protein targeting in living cells by transfection, fluorescent fusion protein expression, and vital staining

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1 Professor Abby Dernburg page 1 of 7 Fluorescent proteins have only been harnessed as an experimental tool for the last dozen years or so - in 1994, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria was successfully expressed in heterologous (other) organisms, the prokaryote E. coli and the eukaryote C. elegans. Since then GFP has been used in essentially every experimental systems. GFP contains a fluorophore in its core, which is enveloped within a β-barrel structure that shields it from the cellular environment.

2 Professor Abby Dernburg page 2 of 7 GFP and other fluorescent proteins (CFP, YFP, RFP) can be fused to a wide variety of proteins without affecting their function. This enables analysis of gene expression, protein localization and dynamics, degradation, protein-protein interactions, you name it! Fluorescent proteins can even be imaged inside whole mice using special high-sensitivity cameras (enough light gets through the skin). This has made it possible to image complex processes, such as tumor growth and regression in response to therapy, using minimally invasive techniques. One specialized way that cells import molecules from their environment is RECEPTOR-MEDIATED ENDOCYTOSIS. In this process, specific receptors that bind to the target molecule are concentrated at particular sites called coated pits on the cells plasma membrane. The receptors concentrate by associating with a protein called CLATHRIN, which forms a meshwork just under the plasma membrane. This enables the cell to concentrate the extracellular molecules at these sites, which eventually pinch off to form clathrincoated vesicles.

3 Professor Abby Dernburg page 3 of 7 Proteins that are secreted from the plasma membrane or targeted to membrane-bound organelles are synthesized by ribosomes on the surface of the Endoplasmic Reticulum (reticulum means net ). As the proteins are made, they are translocated into this giant organelle - you can think of it as a post office where proteins start their journey to other cellular addresses. Vesicles move from the ER to the Golgi and beyond along microtubules that emanate from the MTOC, near the Golgi. This movement is driven by molecular motors. From the Endoplasmic Reticulum, membranebound vesicles bud off and move to the Golgi apparatus, which is a network of membranebound compartments located close to the cell nucleus. The vesicles fuse with and become part of the Golgi apparatus. There, the proteins are modified and resorted for delivery to other parts of the cell. The eventual destination of a protein is encoded within its amino acid sequence (and therefore within the DNA sequence that determines the amino acid sequence). Sometimes these addresses are encoded within a small stretch of amino acids called a SIGNAL SEQUENCE, but other times the information is distributed in different regions of the protein that come together to form a SIGNAL PATCH when the protein folds up into a 3-D sructure. Signal sequences can often be recognized computationally; signal patches are harder to spot. Many signal sequences will work at different places within the protein (e.g., at the N- terminus, the C-terminus, or internally). Other signal sequences work best at specific locations within the protein. If a protein with a signal sequence is fused to another protein - say GFP - that does not have any specific sorting information, the location of the FUSION PROTEIN will most often be determined by the signal sequence.

4 Professor Abby Dernburg page 4 of 7 After verifying that a construct contains the desired transgene, the DNA is introduced into mammalian cells by TRANSFECTION. In this method, DNA crosses the plasma membrane by endocytosis after cations are added to neutralize the negative charges of the phosphate backbone, which prevent it from crossing membranes. Note that there are other techniques to get DNA into cells, including microinjection, electroporation, and viral infection. Fusion proteins can be introduced into cells by creating transgenes - artificial DNA constructs that encode two (or more) proteins as a contiguous sequence. To be expressed in a specific cell type, the fusion gene must have a promoter that that can be recognized by the RNA polymerase machinery in that cell type. Other important regulatory information includes a polyadenylation sequence (this ensures that the mrna is polyadenylated after it is transcribed, which is important for its stability. In addition to these transcriptional signals, the vector or plasmid that contains the transgene must have sequences that allow it to be replicated and selected for in bacterial cells, to enable it to be amplified and purified. Because transfection is not 100% efficient, cells will show variable expression of fluorescent fusion proteins.

5 Professor Abby Dernburg page 5 of 7 In this lab, we will try to identify the cellular locations of several different GFP fusion proteins by co-staining the transfected cells with fluorescent vital stains that localize to known organelles in living cells. These stains have been chosen to fluoresce at different wavelengths than GFP, so that they can be imaged in the same cells using different filters and compared to the GFP localization. Ceramide should localize to the Golgi apparatus under appropriate conditions Transferrin is taken up by receptor-mediated endocytosis. It should concentrate in the RECYCLING EN- DOSOME before being returned to the cell surface.

6 Professor Abby Dernburg page 6 of 7 Mitotracker Red is not fluorescent by itself. It is membrane-permeant, meaning that it will go through the plasma membrane and into other cellular organelles. Inside the environment of a mitochondrion, it becomes oxidized, which makes it become fluorescent. It also gets trapped in the mitochondrion - it can no longer diffuse out into other parts of the cell. The mechanism by which ER-Tracker concentrates in the Endoplasmic Reticulum is not well understood. We are using ER-Tracker Blue-White DPX, which is excited by UV light and has a blue emission maximum, so you can see it with the same filter sets you have used for Hoechst dye. All of the other vital stains in this lab have red emission maxima, so they can be combined with Hoecst if desired.

7 Analysis of protein targeting in living Professor Abby Dernburg page 7 of 7 The major technical challenges faced in this lab are related to the fact that we are looking at living cells. This means you will need to be conscious about: 1. Photobleaching 2. Cellular autofluorescence (which gets worse as cells start to die). 3. Bleedthrough fluorescence (detection of a fluorescent emission signal using the wrong filter sets) - understand why this happens! 4. Nonspecific labeling - can occur if vital stains are used at the wrong concentrations or the conditions are inappropriate - or cells are dying 5. Data management - you have five different vital stains and four different fluorescent fusion proteins, and (along with your labmates) you should try to record images of all 20 possible combinations - Bleed-through fluorescence can often be differentiated from real signal by its color - this requires your eyes, since the camera doesn t detect color.