Reference: Biol. Bull. 201: 240 –241. (October 2001) Recombinant Globular Tail Fragment of Myosin-V Blocks Vesicle Transport in Squid Nerve Cell Extracts Jeremiah R. Brown, Kyle R. Simonetta, Leslie A. Sandberg, Phillip Stafford 1 , and George M. Langford (Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755) Myosin-V, a calmodulin-binding myosin motor, mediates the movement of vesicles on cortical actin filaments in a variety of cell types. This motor has been shown to transport ER and synaptic vesicles in neurons, melanosomes in melanocytes, and secretory ves- icles and the vacuole in yeast. Recent evidence (1) suggests that the globular tail of myosin-V, which binds to the surface of vesicles (2, 3), 1 Motorola, Inc. Chicago, IL. Figure 1. (A) Western blot analyses of immunoprecipitation (IP) experiments using the GST-MyoV-tail. Clarified squid optic lobe homogenate was Triton X-100 extracted, incubated with the recombinant tail fragment for 2 h at 4°C, and recovered using -GST. (Lane 1) Squid myosin-V enriched fraction (S5B) probed with -P196, a polyclonal squid myosin-V antibody (* denotes band of interest). (Lane 2) IP-GST-MyoV-tail probed with -P196. (Lane 3) IP-GST-MyoV-tail probed with -H2, a monoclonal antibody to the squid kinesin. (Lane 4) IP-GST-MyoV-tail probed with -GST antibody. (Lane 5) Purified GST-MyoV-tail probed with -GST. (B) Western blot analyses of sucrose vesicle fractions obtained by running clarified squid brain homogenate on a sucrose density gradient (0.3/0.6/1.5 M gradient; vesicle fraction taken from 0.3/0.6 M interface). (Lane 1) Vesicle fraction probed with -QLLQ, a polyclonal antibody to the squid myosin-V tail. (Lane 2) Vesicle fraction probed with -H2. (C) Western blot analyses of vesicle fraction incubation GST-MyoV-tail. The vesicle fraction, prepared by resuspending vesicular pellet from clarified squid brain homogenate, was incubated for 2 h at 4°C with the GST-MyoV-tail. The control was incubated only with buffer. The supernatant and vesicle pellet were analyzed by western blot analyses. (Lane 1) Control supernatant probed with -QLLQ. (Lane 2) GST-MyoV-tail incubation supernatant probed with -QLLQ. (Lane 3) Control pellet probed with -QLLQ. (Lane 4) GST-myosin-V tail incubation probed with -QLLQ. (Lane 5) GST-MyoV-tail incubation pellet probed with -GST. (Lane 6) Purified GST-MyoV-tail probed with -GST. (D) GST-MyoV-tail inhibition experiments. GST-MyoV-tail was added to squid giant axon extracts at time zero. GST was added to the control. Vesicles moving/field/minute (motile activity) was measured for the control at 15 min. Motile activity was measured for the GST-MyoV-tail at 45 min. Each measured for concentrations of 0.25 mg/ml and 0.5 mg/ml. (E) The motile activity at each GST-MyoV-tail concentration compared with the control is plotted as percent (%) inhibition. Percent inhibition determined by comparing 15-minute control time point with the 45-minute experimental time point. 240 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS