Biology of Membrane Trafficking and Autophagy

Q 1: In the image, Atg9 (green) vesicle and an ER marker are seen together. Based on your experience analyzing similar data and your understanding of the biology of membrane trafficking and autophagy, answer the following questions.

  1. Given the pattern observed, explain whether you can conclude that these cells are undergoing macro autophagy and why or why not.
  2. Identify the steps common to integral membrane proteins needed to get Atg9 into the secretory pathway as well as the unique elements relates to its membrane topology and its trafficking.
  • Address the unique positioning of Atg9 membranes in the example image. Describe the subcellular elements thought to position Atg9 membranes and how they impact your thinking regarding septin diffusion barriers
  1. What major differences in Atg9GFP localization do you predict in atg1(delta) strain treated with rap. Describe overall impact of the mutation on autophagy based on the biology of the pathway. Contrast your prediction with the image provided; what would be different or the same?

 
Q 2: To capture FM 464 staining, the optics of your florescence microscope must be designed to excite the dye and then capture its emitted light. Using your understanding of how a basic florescence microscope is designed, draw and label a diagram of the filters and optics necessary to capture FM 4-64 florescence as shown. Include labels to show each element as well as the specific wavelengths (in nm) of light relevant to each part of the design. List two major advantages of this set up to obtain multi-channel image with resulting image contrast and resolution, as shown in the example.
 
Q 4: Noda and colleagues used biochemical experiments to understand the nature of the Atg9 protein and compartment. Use your understanding of their work and of cell biology more generally to answer the following questions.
 
How did Noda et al. determine that Atg 9 is an integral membrane protein?
Describe the detailed steps in their experiment (e.g. centrifugation, detection, and controls) that positively supported their conclusion that Atg9 is an integral membrane protein. Provide one additional experimental approach that would further support the conclusion that Atg9 is an integral protein.