(no fix, no perm, no antibody) in a transiently transfected culture. As the captions reads: “Note the complex network of actin filaments and stress fibers that extend throughout the cytoplasm and inhabit the cortex in this cell line.” Also examine the example cells displayed at this URL: Similar to the BPAE, this second example of the embryonic rat thoracic aorta A-10 cells are both tri-labeled; however, the molecules stained and/or fluorochomes used are different. In this second case, F-actin is stained with BODIPY FL conjugated to phallacidin. That is, the BODIPY FL fluorophore has spectral properties similar to fluorescein/ FITC (i/e/. blue excitation, green emission) and the phallacidin like phalloidin, is an F-actin stabilizing toxin isolated from the death cap mushroom, Amanita phalloides . Again, note a complex network of actin filaments and stress fibers. Did you see this with the BPAE cells or with your DAPI-RPH-stained cells? Rather than DAPI or Propidium Iodide, DNA is stained with the red- absorbing / orange emitting dye TO-PRO-3. Finally while no tubulin is stained, the third label MitoTracker Orange CMTMRos with a yellow emission targets the mitochondria. Now it is your turn, examine more beautiful pictures of labeled fibroblasts and myoblasts at . html Select one of the 20 representative pictures of the A-10 cell line and briefly compare your selection with your cheek cells and the BPAE cells. How it is similar or different based on the molecules examined, the fluorochrome used and the final composite image obtained? Note, as in the two examples above, many of these pictures show the actin cytoskeleton is some color other than RED Page 5 of 6
EXERCISE 1 Page 6 of 6 (as we saw with Texas Red-Phalloidin) so be careful in your discussion. Explicitly discuss with specific reference to your results and figures (sketches, digital images, etc.) how the actin cytoskeletal network compares among the myoblasts (muscle stem cells), the BPAE (endothelial) and your cheek cells (epidermis). The function of which cell type (myoblast, BPAE, or cheek) do you think would be most significantly affected by the lack of actin ? Support your reasoning. (Hint: see the paper Nemaline Myopathy Caused by Absence of α-Skeletal Muscle Actin. Nowak, et al., 2006.) 3 points 15) The remaining question is also to be answered individually and submitted to your LI separately by each group member. Be sure to include your name, uteid and unique number in the header. Examine the first Selected References on Actin: Cofilin cooperates with fascin to disassemble filopodial actin filaments (PDF) Note you do not have to read the entire paper to successfully answer this question.
- Spring '08
- Bone marrow, digital Images, BPAE