Structure And Function Of Unconventional Myosins and CARMIL Proteins

最近的溶液研究表明，从小鼠和阿米巴CARMIL中分离的CAH 3结构域在加入到先前用加帽蛋白（CP）加帽的肌动蛋白丝中时，能够快速有效地恢复肌动蛋白聚合。 为了直接证明这种假定的去帽活性，我们使用TIRF显微镜在添加小鼠CARMIL-1（mCAH 3）的CAH 3结构域之前和之后观察了单个CP帽化的肌动蛋白丝。 mCAH 3的添加快速恢复了单个封端长丝的聚合，与去封端一致。 为了验证去帽化，用标记有mGFP的重组小鼠CP对细丝加帽。 在mCAH 3添加后聚合的恢复之前，mGFP-CP立即从细丝末端完全解离，证实了CAH 3驱动的去帽机制。 定量分析表明，未加帽的肌动蛋白丝的百分比随着mCAH 3浓度的增加而增加，在250 nM mCAH 3时达到最大值90%。 此外，mCAH 3添加和脱帽之间的时间间隔随着mCAH 3浓度的增加而减少，其中CP在倒刺末端的平均半衰期从没有mCAH 3的30分钟减少到具有饱和量的mCAH 3的10秒。 在这些基于TIRF的测定中，含有消除其与游离CP的紧密结合的单点突变（R993 E）的mCAH 3完全没有去帽活性。 最后，使用mGFP标记的mCAH 3，我们获得了直接的证据，证明CAH 3和CP的复合物对倒刺末端具有小但可测量的亲和力，如从动力学建模推断的。 我们的结论是，CAH 3结构域的CARMIL，大概是完整的分子，以及具有uncap CP-帽肌动蛋白丝的能力。 这种活性可能会沿着从头成核和细丝切断，在体内产生游离倒刺末端，并且可能至少部分导致CP在细胞内倒刺末端的半衰期较短（JCB，2006）。 我们的研究结果与最近的一份报告形成对比，该报告称，PIP 2也被认为是从溶液研究中去除CP帽的细丝，但当通过TIRF显微镜检查时似乎没有这样做（JBC，2007）。 网骨藻中的管泡收缩泡（CV）复合体与富含肌动蛋白的皮层有广泛的联系，并沿着运动。 在这里，我们表明，V型肌球蛋白myoJ的目标CV膜，myoJ空细胞表现出增加的敏感性，低渗条件下，CV膜从皮层的戏剧性损失，和CV膀胱无法进行tuberance以下水排放。 用GJP标记的myoJ补充myoJ-细胞完全挽救了CV膜的皮质结合和CV小管的运动性的缺陷。 与myoJ版本的互补，要么走得很慢，要么采取更短的步骤，导致在两种情况下的皮质CV膜分布的救援，但救援的肾小管仅在步长突变体的情况下，和肾小管在这种情况下，以其正常速度的一半移动。 最后，在myoJ细胞中观察到的MTOC周围CV膜的稳态积累迫使我们以可以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微管不仅在膜平面内的皮层肌动蛋白上移动，而且还沿皮层和MTOC之间的微管双向沿着移动。 因此，除了myoJ在驱动CV膜的皮质联合和运动中的作用外，它还与正和负末端定向微管马达合作，以驱动网骨藻中CV复合物的适当分布和动力学。

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.