Abstract
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.
Highlights
The enzymology, assembly and regulation of Acanthamoeba myosin-I and myosin-II have been studied in detail (Sinard and Pollard, 1989; Brzeska et al, 1992; Ostap and Pollard, 1996)
Fluorescent antibody staining of fixed cells showed that myosin-II forms particles of two sizes (Yonemura and Pollard, 1992) corresponding to mini-filaments of eight molecules and thick filaments formed by lateral association of mini-filaments (Sinard and Pollard, 1989)
Myosin dynamics have been documented in animal cells (Wei and Adelstein, 2000; Buss et al, 2001; Tang and Ostap, 2001), Dictyostelium (Moores et al, 1996; Zang and Spudich, 1998; Yumura, 2001; Neujahr et al, 1997) and yeast (Bezanilla et al, 2000; Lee et al, 2000) by expressing myosin fusion proteins tagged with green fluorescent protein (GFP)
Summary
The enzymology, assembly and regulation of Acanthamoeba myosin-I and myosin-II have been studied in detail (Sinard and Pollard, 1989; Brzeska et al, 1992; Ostap and Pollard, 1996). Myosin dynamics have been documented in animal cells (Wei and Adelstein, 2000; Buss et al, 2001; Tang and Ostap, 2001), Dictyostelium (Moores et al, 1996; Zang and Spudich, 1998; Yumura, 2001; Neujahr et al, 1997) and yeast (Bezanilla et al, 2000; Lee et al, 2000) by expressing myosin fusion proteins tagged with green fluorescent protein (GFP) Such GFP fusion proteins can take the place of wild-type Dictyostelium myosin-II (Moores et al, 1996), yeast myosinII (Bezanilla et al, 2000) and yeast myosin-I (Lee et al, 2000)
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