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，当将肌动蛋白聚合添加到先前用上限蛋白（CP）上盖的肌动蛋白丝中时，从小鼠和acanthamoeba Carmil中分离出的CAH3结构域，可恢复肌动蛋白的聚合。为了直接证明这种推定的解抗活性，我们在使用TIRF显微镜从小鼠Carmil-1（MCAH3）添加CAH3域之前和之后观察到了单个CP抑制的肌动蛋白丝。 MCAH3的添加迅速恢复了单个上限细丝的聚合，与脱覆一致。为了验证解蛋白，用重组小鼠CP限制了细丝，用MGFP标记。在添加MCAH3上的聚合恢复之前，MGFP-CP从细丝端完全解离，从而确认了CAH3驱动的解染机制。定量分析表明，随着添加的MCAH3浓度的增加，未覆盖的肌动蛋白丝的百分比增加，在250 nm MCAH3时最多达到90％。此外，随着MCAH3浓度的增加，MCAH3添加和Uncapping之间的时间间隔减少了，钢筋末端的CP平均半衰期从30分钟从没有MCAH3的30分钟下降到10秒，而MCAH3饱和。 MCAH3包含一个点突变（R993E），它消除了其与游离CP的紧密结合完全没有这些基于TIRF的测定中的未覆盖活性。最后，使用用MGFP标记的MCAH3，我们获得了直接的证据，表明CAH3和CP的复合物对带状端具有较小但可测量的亲和力，这是根据动力学建模推断的。我们得出的结论是，Carmil的孤立CAH3结构域，并且大概是完整的分子，它具有开除CP抑制肌动蛋白丝的能力。这种活性可能会与从头核和细丝切断，体内的自由带末端的产生，并且至少在细胞内部的CP半衰期中可能是造成的（JCB，2006年）。我们的结果与最近的一份报告形成鲜明对比的是，从解决方案研究到UNCAP CP覆盖的细丝的PIP2在通过TIRF显微镜检查时似乎没有这样做（JBC，2007）。 dictyostelium中的肾小管收缩液泡（CV）复合物与富含肌动蛋白的皮质沿着运动表现出广泛的关联和运动性。在这里，我们表明，V型肌球蛋白MyoJ靶向CV膜，并且MyoJ Null细胞对低渗透条件的敏感性增加，皮质中CV膜的急剧损失以及CV Bladders无法在水排出后butegangue的小管。用GJP标记的MyoJ互补的肌j-细胞充分挽救了CV膜皮质关联和CV小管运动性的缺陷。在两种情况下，与Myoj的版本相互互补，或者采取较短的步骤会导致皮质CV膜分布的挽救，但仅在步骤尺寸突变体的情况下挽救管道，而这种情况下的小管以正常速度移动。最后，在MyoJ细胞中看到的MTOC周围的CV膜的稳态积累迫使我们以方式可视化CV膜动力学，这可以突出可能突出可能的微管依赖性运动。这些图像表明，CV小管不仅在膜平面上的皮质肌动蛋白上移动，而且在皮质和MTOC之间的微管方向上进行双向移动。因此，除了MyOJS在驱动CV膜的皮质关联和运动中的作用外，它还与Plus和Minus终端导向的微管电机合作，以驱动Dictyostelium中CV复合物的适当分布和动力学。

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。