Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.
Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.
复制标题
肌球蛋白18A与非肌肉肌球蛋白2结合形成混合的双极丝。
DOI：
10.1016/j.cub.2015.02.012
复制
发表时间：
2015-03-30
期刊：
影响因子：
--
通讯作者：
Sellers JR
中科院分区：
文献类型：
--
作者：
Billington N;Beach JR;Heissler SM;Remmert K;Guzik-Lendrum S;Nagy A;Takagi Y;Shao L;Li D;Yang Y;Zhang Y;Barzik M;Betzig E;Hammer JA 3rd;Sellers JR
18类肌球蛋白与常规的2类非肌肉肌球蛋白（NM2）关系最为密切。令人惊讶的是，果蝇、小鼠和人类肌球蛋白18A（M18A）的纯化头部结构域缺乏肌动蛋白激活的ATP酶活性以及转运肌动蛋白丝的能力，这表明M18A在体内的功能不依赖于内在的运动活性。M18A具有除2类肌球蛋白之外所有肌球蛋白中最长的卷曲螺旋结构，这表明它可能形成类似于常规肌球蛋白的双极丝。为了探究这种可能性，我们使用杆状病毒/Sf9系统表达并纯化了全长小鼠M18A。在测试的任何条件下，M18A都没有形成大的双极丝。相反，M18A形成了一种约65纳米长的双极结构，两端各有一个头部。重要的是，当NM2在M18A存在的情况下聚合时，这两种肌球蛋白形成了混合双极丝，这通过共沉降、电子显微镜和单分子成像得到了证明。此外，使用荧光标记蛋白对NM2和M18A进行的超分辨率成像以及对内源蛋白的免疫染色表明，NM2和M18A一起存在于活细胞内的单个丝中。总之，我们的体外和活细胞成像数据有力地表明，M18A与NM2共同组装成混合双极丝。M18A可以调节这些丝的生物物理特性，并且凭借其额外的N端和C端结构域，确定丝的定位和/或分子相互作用。鉴于NM2具有众多基本的细胞和发育作用，我们的结果具有深远的生物学意义。
Class 18 myosins are most closely related to conventional class 2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, arguing that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled-coil of any myosin outside of the class 2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any conditions tested. Instead, M18A formed a ~65 nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments, and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far reaching biological implications.
登录
查看更多内容
DOI：
10.1074/jbc.m110.212290
发表时间：
2011-06-17
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Heissler SM;Manstein DJ
通讯作者：
Manstein DJ
影响因子：
3.7
作者：
Hodges JL;Newell-Litwa K;Asmussen H;Vicente-Manzanares M;Horwitz AR
通讯作者：
Horwitz AR
DOI：
10.1038/nrm2786
发表时间：
2009-11
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1074/jbc.m111.218669
发表时间：
2011-06-17
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Guzik-Lendrum S;Nagy A;Takagi Y;Houdusse A;Sellers JR
通讯作者：
Sellers JR
DOI：
10.1073/pnas.1007025107
发表时间：
2010-12-07
影响因子：
11.1
作者：
Ricketson, Derek;Johnston, Christopher A.;Prehoda, Kenneth E.
通讯作者：
Prehoda, Kenneth E.