You identify a mutant that blocks melanosome movement. You rule out any defects in melanosome formation, dynein function or cell signaling. You wonder whether kinesin-2 or the cytoskeleton itself could be disrupted. What is the best way you could determine if and how the subcellular organization of the cytoskeleton is specifically affected for kinesin-2 function in the mutant cells? a. Perform cell fractionation and then use immunoblots with an antibody against alpha-tubulin to detect microtubules, gamma-tubulin to see centrosomes, and +TIP proteins to see plus (+) ends of microtubules.
b. Perform immunofluorescence with antibodies against alpha-tubulin to detect microtubules, gamma-tubulin to see centrosomes, and + TIP proteins to see plus (+) ends of microtubules.
c. Perform a bead assay, using bead coated with kinesin-2 on slides coated with cell lysates from either wildtype or mutant cells.
d. Perform immunofluorescence to detect F-actin and antibodies to specific capping proteins to detect plus (+) and minus (-) ends of microfilaments.
e. Perform a FRAP assay of fluorescently-labeled microtubules.