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doi: 10.1242/10.1242/jcs.00159

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Intracellular localization and dynamics of myosin-II and myosin-IC in live Acanthamoeba by transient transfection of EGFP fusion proteins

Hyun-Hee Kong^1,* and Thomas D. Pollard^2,

¹ Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
² Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA
^* Present address: Department of Parasitology, Kyungpook National University School of Medicine, Taegu 700-422, Korea

Author for correspondence (e-mail: thomas.pollard{at}yale.edu)

Accepted 4 September 2002

We developed a reliable method for transient transfection of Acanthamoeba using Superfect (Qiagen) and a vector with the Acanthamoeba ubiquitin promoter and enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was 3% for profilin-I-EGFP and EGFP-myosin-II tail, and less than 0.5% for larger constructs such as full length myosin-II or myosin-IC. Profilin-I-EGFP was distributed throughout the cytoplasm as observed previously with rhodamine-labeled profilin, while EGFP alone accumulated in the nucleus. EGFP fused to full length myosin-II or to the C-terminal 256 residues of the myosin-II tail concentrated in fluorescent spots similar to thick filaments and minifilaments identified previously in fixed cells with fluorescent antibodies. Thick filaments were located in the dorsal cytoplasm and along the lateral margins of the back half of the cell. Thick filaments formed behind the leading edge and moved continuously towards the rear of the cell, where they disassembled. If phosphorylation of the myosin-II heavy chain was prevented by mutation of all three phosphorylated serines to alanine, thick filaments of unphosphorylated myosin-II accumulated around vesicles of various sizes. EGFP-myosin-IC was spread throughout the cytoplasm but concentrated transiently around contractile vacuoles and macropinocytosis cups providing that the construct included both the head and a tail with the SH3 domain.

Key words: Myosin, Motility, Microscopy, Green fluorescent protein, Transfection

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