MCDB 165a Take home quiz 5 Rizzo et al (2013), Journal of Cell Biology. 1. When the MANIFM is in the pellet form, it means that it is a polymer and when the MANIFM is in the supernatant form, it is a monomer. The polymer and monomer forms of MANIFM play different roles in the model that is being presented. Therefore, it is important to know where the two different forms of the MANIFM are present in the Golgi complex. The main result of the experiment was that when the AP was washed out, the MANIFM polymerized and when the AP was added back into the MANIFM, they were depolymerized back into monomers. This experiment therefore clearly shows the role that AP plays in the polymerization and depolymerization of the MANIFM. 2. Since cycloheximide is a translational inhibitor, it inhibits the synthesis of new proteins. In the results for experiments shown in Figure 2, the MANIFM is being observed to see where it localizes in the Golgi stack when AP is present. Since the proteins are being tracked, it would be hard to track the same proteins over a period of time since new proteins would continue to be synthesized and transported into the Golgi from the ER if cycloheximide is not used. Therefore, the EM images would have a lot more MANIFM present in them and it would be difficult to exactly measure or trace the movements of the resident protein. 3. Yes, these experiments can be done in live cells using fluorescent proteins. Therefore, the immunostaining can no longer be used. In order to use fluorescent proteins, the MANIFM would have the GFP gene incorporated into the MANIFM proteins domain. The GM130 can have the CFP gene, which is a blue fluorescent protein. Further, the TGN46 can have the mCherry gene in its domain. Confocal microscopy can be used again to view and image the lives cells. Altogether, this should produce a live image very similar to the one that is already in the paper. 4. The localization of MANIFM was examined with respect to COP1 because COP1 is a protein that moves from trans to cis. The vesicles and tubules that transport the MANIFM are also coated with COP1 to help transport them back to the cis part of the Golgi. Therefore, visualizing and quantifying the number of COP1 present can confirm the transport of the MANIFM back to the cis end of the Golgi in a retrograde pathway. The MANIFM in the vesicles is mostly likely going to be monomeric because the polymeric MANIFM would be too large and wouldnt be able to enter the vesicle or tubules.