Optimized filter set and viewing conditions for the S65T mutant of GFP in living cells

Investigators are increasingly taking advantage of the autonomous fluorescence from the green fluorescent protein (GFP) to detect the cellular and subcellular location of GFP-fusion proteins within living cells (3,8–10,13,14). Work with these fusion constructs has shed new light on the kinetics underlying fundamental biological processes such as mitosis (9). In an attempt to study how a novel rat brain kinesin-related motor protein (rbKRP1) functions within living cells, we have transfected BHK and PC12 cells with a cDNA encoding the serine 65 ® threonine 65 (S65T) GFP mutant (6) fused to the amino terminus of rbKRP1 (S65T GFP–rbKRP1). We use the S65T GFP mutant since it emits four times as much light as the wild-type GFP (6), and it can enhance our ability to detect localized kinesin motor molecules

Association of a Nonmuscle Myosin II with Axoplasmic Organelles

Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor