J Comprehensible Results

Prosser, D. C., et al.
"FFAT Rescues VAPA-Mediated Inhibition of ER-to-Golgi
Transport and VAPB-Mediated ER Aggregation."
Journal of Cell Science, vol. 121, no. 18, 19 June 2008, pp. 3052-3061.,
doi:10.1242/jcs.028696.

(Translated by Sohail Syed)

Time Course Immunofluorescence of VSVG with relationship to the FFAT motif

Motivation The motivation behind this experiment was to look for whether or not proteins folded correctly and whether or not those transmembrane proteins diffused from the ER to the Golgi and from there, to the membrane. The different time stamps were taken to allow experimenters to see when the transition would occur. This experiment also allows for checking to see how the FFAT motif's expression helps with diffusion. Procedure VSVG is used for this experiment as it is synthesized by the ER and can be tagged for Immunofluorescence. Also it is temperature sensitive meaning it can be stopped from exiting the ER at will with a temperature change. For the experiment without the FFAT motif: 1. Grow VAP cells at 39°C for 16 hours in paraformaldehyde 2. Increase temperature to 42°C for 6 hours 3. Add fluorescence flags for VSVG 4. Add VSVG flag antibodies 5. Drop cells to 32°C to see transport of proteins 6. Immunofluorescence experiment 7. Image at 0 min, 30 min, 60 min, and 90 min
For experiment with FFAT motif: 1. Grow VAP with addition of FFAT motifcells at 39°C for 16 hours in paraformaldehyde 2. Increase temperature to 42°C for 6 hours 3. Add fluorescence flags for VSVG 4. Add VSVG flag antibodies 5. Drop cells to 32°C to see transport of proteins 6. Immunofluorescence experiment 7. Image at 0 min, 30 min, 60 min, and 90 min Results Control Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane Control + FFAT Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane VAPA WT Blocking of transport into membranes 30 min in ER 60 min in Golgi and some in the ER 90 min in Golgi and some in the ER VAPA WT + FFAT Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane VAPA P56S Blocking of transport into membranes 30 min in ER 60 min in Golgi and some in the ER 90 min in Golgi and some in the ER VAPA P56S + FFAT Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane VAPB WT Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane VAPB WT + FFAT Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane VAPB P56S Blocking of transport into Golgi ER membrane has aggregates messing up structure of membranes 30 min in ER 60 min stuck in ER 90 min stuck in ER VAPB P56S + FFAT Transport of proteins is normal 30 min in ER 60 min in Golgi and some in the ER 90 min in Membrane VAPB P56S has increased immobile obstacles leading to less ER transport of transmembrane proteins meaning there are more ER aggregates FFAT motif helps in protein folding allowing less aggregation with the ER and flow through the ER to the Golgi to the Cytosol
FRAP (Fluorescence Recovery After Photobleaching) of Immunofluorescence of VSVG Cells Motivation The motivation of this experiment was to check if diffusion of proteins is still working in relation to VAPs and FFAT motif and also to check to see which of the VAPs have more immobile obstacles, which are obstructions to proteins folding and moving out of the ER. Procedure Steps for FRAP analysis: 1. Image cell before bleaching 2. Hit a region of a cell with a laser bleaching the fluorescence protein 3. Image cell every 20 sec within a 10 min period 4. Graph recovery values 5. Take the linear slope of the graph from 0-2 min Results
Control Recovery is a normal rate Control + FFAT Recovery is a normal rate VAPA WT Recovery is at a slower rate VAPA WT + FFAT Recovery is a normal rate VAPA P56S Recovery is at a slower rate VAPA P56S + FFAT Recovery is a normal rate VAPB WT Recovery is a normal rate VAPB WT + FFAT Recovery is a normal rate VAPB P56S Recovery is at a slower rate VAPB P56S + FFAT Recovery is a normal rate Over-expression of VAPA and VAPB P56S leads to a slower recovery rate Concentration of immobile obstacles increase FFAT rescues recovery rate and decreases concentration of immobile obstacles