Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
J Biol Chem. 2010 Nov 19;285(47):36577-85. doi: 10.1074/jbc.M110.142638. Epub 2010 Sep 7.
A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and "zebra-striped" appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter- and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility.
一种新的肌动球蛋白调节形式已经被提出，即新的肌动蛋白丝聚合调节顶复门原生动物寄生虫的运动。在体内和体外寄生虫 F-肌动蛋白非常短且不稳定，但细丝动力学的结构基础和细节仍不清楚。在这里，我们表明，通过将未标记的兔骨骼肌肌动蛋白（RS-actin）聚合到短的罗丹明-鬼笔环肽稳定的恶性疟原虫肌动蛋白 I（Pf-actin）细丝的两端，可以获得长的肌动蛋白丝。退火后，通过荧光显微镜观察到长微米的杂交细丝，具有“斑马条纹”外观，并且足够稳定，可以在滑行细丝测定中移动肌球蛋白 II 类电机。使用负染色电子显微镜，我们发现用 Jasplakinolide（JAS）稳定的纯 Pf-actin 也形成长丝，在长度上与 RS-actin 丝无法区分，并且足够长，可以进行结构表征。为了在近生理条件下比较水溶液中的结构，我们通过原子力显微镜（AFM）对 Pf-actin 和 RS-actin 细丝进行成像。我们发现 Pf-actin 的单体堆积与 RS-actin 明显不同，使得 Pf-actin 细丝的双螺旋螺距大 10%。与 RS-actin 相比，寄生虫中的亚基旋转角度较大，可以解释我们的结果。使用高分辨率肌动蛋白丝模型对 AFM 数据进行建模支持我们对数据的解释。这里报道的结构差异可能是由于相间和链内接触较弱的结果，并且可能对细丝动力学的差异和寄生虫运动的调节至关重要。
