Structure And Function Of Unconventional Myosins and CARMIL Proteins

非常规肌球蛋白和 CARMIL 蛋白的结构和功能

基本信息

批准号：
7734940
负责人：
JOHN A HAMMER
金额：
$ 200.19万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734940
关键词：
Acanthamoeba Actins Address Affinity Amoeba genus Aspergillus Automobile Driving Binding Biochemistry Biological Biological Assay Biological Models Bladder Cell physiology Cells Class Complex Condition Defect Dictyostelium Dissociation Exhibits Filament Generations Goals Half-Life Image Individual Kinetics Lymphocyte Measurable Membrane Microfilaments Microtubule-Organizing Center Microtubules Modeling Motor Movement Mus Myosin ATPase Myosin Type V Neurons Null Lymphocytes Phosphatidylinositol 4,5-Diphosphate Point Mutation Proteins Recombinants Regulation Reporting Role Signal Pathway Solutions Speed Structure Thinking Total Internal Reflection Fluorescent Vacuole Walking Water Work actin capping protein base cell motility cell type in vivo melanocyte mutant polymerization restoration single molecule size structural biology time interval

项目摘要

Recent solution studies have shown that the isolated CAH3 domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with Capping Protein (CP). To demonstrate this putative uncapping activity directly, we observed single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3) using TIRF microscopy. Addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with mGFP. Restoration of polymerization upon mCAH3 addition was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of uncapped actin filaments increased with increasing concentration of mCAH3 added, reaching a maximum of 90% at 250 nM mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased with increasing concentration of mCAH3, with the average half-life of CP at the barbed end decreasing from 30 minutes without mCAH3 to 10 seconds with saturating amounts of mCAH3. mCAH3 containing a single point mutation (R993E) that abrogates its tight binding to free CP was totally devoid of uncapping activity in these TIRF-based assays. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CAH3 and CP has a small but measurable affinity for the barbed end, as inferred from kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL, and presumably the intact molecule as well, possesses the ability to uncap CP-capped actin filaments. This activity may drive, along with de novo nucleation and filament severing, the generation of free barbed ends in vivo, and may be responsible at least in part for the short half-life of CP at the barbed end inside cells (JCB, 2006). Our results contrast with a recent report that PIP2, which was also thought from solution studies to uncap CP-capped filaments, does not appear to do so when examined by TIRF microscopy (JBC, 2007). The tubulovesicular contractile vacuole (CV) complex in Dictyostelium exhibits extensive association with and motility along the actin-rich cortex. Here we show that the type V myosin myoJ targets to CV membranes, and that myoJ null cells exhibit increased sensitivity to hypo osmotic conditions, a dramatic loss of CV membranes from the cortex, and an inability of CV bladders to undergo tubulation following water discharge. Complementation of myoJ- cells with GJP-tagged myoJ fully rescues the defects in cortical association of CV membranes and motility of CV tubules. Complementation with versions of myoJ that either walk very slowly or take shorter steps results in rescue of cortical CV membrane distribution in both cases, but rescue of tubulation only in the case of the step size mutant, and the tubules in this case move at half their normal speed. Finally, a steady state accumulation of CV membranes around the MTOC seen in myoJ- cells forced us to visualize CV membrane dynamics in way that could highlight possible microtubule-dependent movements. These images revealed that CV tubules move not only on cortical actin in the plane of the membrane, but bi directionally along microtubules between the cortex and the MTOC as well. Therefore, in addition to myoJs role in driving the cortical association and motility of CV membranes, it cooperates with plus and minus end-directed microtubule motors to drive the proper distribution and dynamics of the CV complex in Dictyostelium.

最近的解决方案研究表明，当将来自小鼠和棘阿米巴 CARMIL 的分离 CAH3 结构域添加到先前用加帽蛋白 (CP) 封端的肌动蛋白丝中时，可以快速有效地恢复肌动蛋白聚合。为了直接证明这种推定的脱帽活性，我们使用 TIRF 显微镜观察了添加来自小鼠 CARMIL-1 (mCAH3) 的 CAH3 结构域之前和之后的单个 CP 加帽肌动蛋白丝。 mCAH3 的添加迅速恢复了单个加帽丝的聚合，与脱帽一致。为了验证脱帽情况，用带有 mGFP 标记的重组小鼠 CP 对细丝进行加帽。添加 mCAH3 后聚合恢复立即发生，mGFP-CP 从丝末端完全解离，证实了 CAH3 驱动的脱帽机制。定量分析表明，随着 mCAH3 添加浓度的增加，未加帽的肌动蛋白丝的百分比增加，在 250 nM mCAH3 时达到最大值 90%。此外，随着mCAH3浓度的增加，mCAH3添加和开盖之间的时间间隔减少，带刺端的CP的平均半衰期从没有mCAH3的30分钟减少到mCAH3饱和量的10秒。在这些基于 TIRF 的测定中，含有单点突变 (R993E) 的 mCAH3 消除了与游离 CP 的紧密结合，完全没有脱帽活性。最后，使用带有 mGFP 标记的 mCAH3，我们获得了直接证据，表明 CAH3 和 CP 的复合物对有刺末端具有较小但可测量的亲和力，如动力学模型推断的那样。我们得出的结论是，CARMIL 的分离的 CAH3 结构域（可能还有完整的分子）具有解开 CP 加帽肌动蛋白丝的能力。这种活性可能与从头成核和细丝切断一起驱动体内游离倒刺末端的产生，并且可能至少部分地导致细胞内倒刺末端的 CP 半衰期较短（JCB，2006）。我们的结果与最近的一份报告形成对比，PIP2 也被认为可以从溶液研究中解开 CP 封端的细丝，但在 TIRF 显微镜检查时似乎没有这样做（JBC，2007）。盘基网柄菌中的管泡收缩液泡 (CV) 复合体与富含肌动蛋白的皮层表现出广泛的关联和运动。在这里，我们表明，V 型肌球蛋白 myoJ 靶向 CV 膜，并且 myoJ 无效细胞对低渗透压条件的敏感性增加，皮质中的 CV 膜急剧丧失，并且 CV 膀胱在排水后无法进行管状化。用 GJP 标记的 myoJ 补充 myoJ 细胞可以完全挽救 CV 膜皮质关联和 CV 小管运动的缺陷。与行走速度非常慢或步数较短的 myoJ 版本互补会导致两种情况下皮质 CV 膜分布的挽救，但仅在步长突变体的情况下才能挽救管状结构，并且在这种情况下，肾小管的移动速度是其一半正常速度。最后，在 myoJ 细胞中观察到 MTOC 周围 CV 膜的稳态积累迫使我们以能够突出可能的微管依赖性运动的方式可视化 CV 膜动力学。这些图像显示，CV 小管不仅在膜平面上的皮质肌动蛋白上移动，而且还沿着皮层和 MTOC 之间的微管双向移动。因此，除了 myoJ 在驱动皮质关联和 CV 膜运动方面的作用外，它还与正负端定向微管马达配合，驱动盘基网柄菌中 CV 复合体的正确分布和动态。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Melanophilin and myosin Va track the microtubule plus end on EB1.

DOI：
10.1083/jcb.200503028
发表时间：
2005-10-24
期刊：
JOURNAL OF CELL BIOLOGY
影响因子：
7.8
作者：
Wu, Xufeng S;Tsan, Grace L;Hammer, John A 3rd
通讯作者：
Hammer, John A 3rd

The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

DOI：
10.1083/jcb.153.7.1479
发表时间：
2001-06-25
期刊：
The Journal of cell biology
影响因子：
0
作者：
Jung G;Remmert K;Wu X;Volosky JM;Hammer JA 3rd
通讯作者：
Hammer JA 3rd

The cargo-binding domain regulates structure and activity of myosin 5.

DOI：
10.1038/nature04865
发表时间：
2006-07-13
期刊：
NATURE
影响因子：
64.8
作者：
Thirumurugan, Kavitha;Sakamoto, Takeshi;Hammer, John A., III;Sellers, James R.;Knight, Peter J.
通讯作者：
Knight, Peter J.

CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments.

CARMIL 是一种有效的加帽蛋白拮抗剂：鉴定了一个保守的 CARMIL 结构域，该结构域抑制加帽蛋白的活性并解开加帽肌动蛋白丝。