Structure And Function Of Unconventional Myosins

非常规肌球蛋白的结构和功能

• 批准号: 6966861
• 负责人: JOHN A HAMMER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/6966861
• 关键词: Dictyostelium; guanosinetriphosphatases; intracellular transport; laboratory mouse; melanocyte; melanosomes; microtubules; myosins; protein folding; protein isoforms; protein protein interaction; protein structure function; vesicle /vacuole

Microtubule plus end tracking proteins or +TIPS have been implicated in the control of microtubule dynamics, cell polarity, spindle positioning, and organelle: microtubule interactions that precede dynein-dependent organelle motility. We have shown that the distribution of melanosomes in mouse melanocytes is driven by long range, bi-directional, microtubule-dependent movements and local, myosin Va-dependent movements on actin in the periphery. We have also shown that Myosin Va is recruited on to the melanosome surface by a receptor complex containing Rab27a present on the melanosome membrane and melanophilin, which links myosin Va to Rab27a. We now show that GFP-tagged melanophilin also exhibits microtubule plus end tracking behavior. Moreover, GFP-tagged myosin Va can also surf the microtubule plus end and does so in a melanophilin-dependent manner. Finally, alterations in the cellular levels of the +TIP EB1, as well as pull down assays, argue that melanophilin surfs indirectly by hitchhiking on EB1. These results indicate that vertebrate cells have retained in the form of a myosin Va-melanophilin-EB1 complex the connection between microtubule plus ends and F-actin seen in the yeast complex containing Myo2p (a type V myosin), Bim1p (an EB1 homolog), and Kar9p, which links Myo2p to Bim1p. Given melanophilin?s documented role in coupling Rab27a-bearing vesicular cargo to myosin Va, we suggest that the accumulation of melanophilin and myosin Va at the microtubule plus end may serve to focus and facilitate the transfer of melanosomes from microtubules to actin at this location. The contractile vacuole (CV) complex is a specialized intracellular membrane compartment that serves as the osmoregulatory organelle in protozoa. In Dictyostelium, this compartment is composed of an interconnected network of tubules and cisternae or bladders. These membranes accumulate excess water (e.g. rain water) that has entered the cell by osmosis by pumping protons and most likely bicarbonate into their lumen. The resulting ion gradient draws the excess water out of the cytoplasm and into the lumen. The swollen bladders that are generated expel this excess water from the cell through a transient fusion pore in the plasma membrane. The tubules and bladders that comprise the system are highly dynamic and very pleiomorphic, are rapidly interconvertible, and do not mix with the endosomal/lysosomal membrane system or with the plasma membrane during the process of water expulsion. What has emerged from prior studies is a working definition of a CV membrane cycle in Dictyostelium in which swollen, mature bladders contact the fusion pore in the plasma membrane, water is discharged from the cell, the collapsed bladder membrane folds up into a tight knot immediately under the plasma membrane, this knot of membrane rapidly transforms into tubules that radiate out across the actin rich cortex, and these tubules fuse with each other and with immature bladders during the filling phase to create new mature bladders. These cortical events are seen best in time lapse confocal images of the ventral surface of adherent cells, as this configuration places a large area of the plasma membrane and subjacent actin-rich cortex within a single focal plane. The close association of CV membranes with the actin-rich cortex, and the dramatic motility of CV tubules along the cortex have led to the suggestion that CV membranes recruit some type of myosin. We now show that the Dictyostelium type V myosin myoJ is targeted to CV membranes and is responsible for their steady state association with the actin-rich cortex. Moreover, we show that myo J drives the tubulation of collapsed bladder membranes along the cortex following water discharge. Finally, the steady state accumulation of CV membranes around the MTOC seen in myoJ null cells forced us to visualize CV membrane dynamics in the middle off the cell as well as along its ventral surface. These images revealed that the tubules emanating from collapsed bladders move not only on actin in the plane of the membrane but bidirectionally along microtubules between the cortex and the microtubule organizing center (MTOC) adjacent to the nucleus. From this we conclude that myoJ cooperates with plus and minus end-directed microtubule motors to drive the proper distribution and function of the CV complex in Dictyostelium. The coat color phenotypes of dilute (myosin Va-), ashen (Rab27a-) and leaden (melanophilin-) mice are identical and nonadditive because the absence of any one of these gene products completely abrogates the interaction of melanosomes with the actin cytoskeleton. We have begun to characterize dilute suppressor (dsu), an extragenic suppressor of dilute, ashen and leaden, which our collaborators recently identified by backcrossing and bac transgene rescue. Dsu encodes a novel, highly-charged protein of ~22 kDa. Consistent with the deletion mutation present in the dsu allele, dsu melanocytes are devoid of the dsu protein (dsup). Therefore, it is the loss of expression of dsup that causes rescue. Dilute melanocytes that are also homozygous dsu show a significant spreading of melanosomes throughout the cytoplasm, which most likely explains the restoration of coat color. But melanosomes in these cells never concentrate in dendritic tips, presumably because this process is strictly myosin Va-dependent. Consistently, overexpression of GFP-tagged dsup in dilute/dsu melanocytes causes melanosomes to once again cluster in the cell center. Moreover, these cells show an almost exclusive colocalization of GFP-tagged dsup with melanosomes. Melanosome targeting of dsup requires the addition of multiple palmitates at a cluster of six cysteines located near its N-terminus, suggesting that dsup may be a component of a specialized melanosomal membrane micro domain. We are now characterizing the nature of microtubule-based melanosome movements in melanocytes lacking and over expressing dsup to determine if dsup causes rescue by effecting the balance between plus and minus end-directed movements of melanosomes on microtubules.

微管加末端追踪蛋白或+TIPS 参与微管动力学、细胞极性、纺锤体定位和细胞器的控制：微管相互作用先于动力蛋白依赖性细胞器运动。我们已经证明，小鼠黑素细胞中黑素体的分布是由远距离、双向、微管依赖性运动和外周肌动蛋白上的局部、肌球蛋白 Va 依赖性运动驱动的。我们还表明，肌球蛋白 Va 通过包含黑素体膜上存在的 Rab27a 和亲黑素的受体复合物募集到黑素体表面，该受体复合物将肌球蛋白 Va 与 Rab27a 连接。我们现在表明 GFP 标记的亲黑素也表现出微管加末端跟踪行为。此外，GFP 标记的肌球蛋白 Va 也可以在微管正端冲浪，并且以亲黑素依赖性方式进行。最后，+TIP EB1 细胞水平的变化以及下拉分析表明，亲黑素通过搭 EB1 的便车间接冲浪。这些结果表明，脊椎动物细胞以肌球蛋白 Va-亲黑素-EB1 复合物的形式保留了微管正端和 F-肌动蛋白之间的连接，这种连接在含有 Myo2p（V 型肌球蛋白）、Bim1p（EB1 同源物）的酵母复合物中可见。和 Kar9p，它将 Myo2p 链接到 Bim1p。鉴于亲黑素在将带有 Rab27a 的囊泡货物与肌球蛋白 Va 偶联中发挥的作用，我们认为亲黑素和肌球蛋白 Va 在微管正端的积累可能有助于集中并促进黑素体从微管转移到该位置的肌动蛋白。 收缩液泡（CV）复合体是一种特殊的细胞内膜室，充当原生动物的渗透调节细胞器。在盘基网柄菌属中，该隔室由相互连接的小管和池或膀胱网络组成。这些膜积聚了多余的水（例如雨水），这些水通过渗透作用通过将质子和很可能是碳酸氢盐泵入其内腔而进入细胞。由此产生的离子梯度将多余的水从细胞质中吸出并进入管腔。产生的肿胀的膀胱通过质膜上的瞬时融合孔将多余的水从细胞中排出。组成该系统的小管和膀胱是高度动态的和非常多形性的，可以快速相互转换，并且在水排出过程中不与内体/溶酶体膜系统或质膜混合。先前的研究得出了盘基网柄菌属CV膜循环的工作定义，其中肿胀、成熟的膀胱接触质膜中的融合孔，水从细胞中排出，塌陷的膀胱膜立即折叠成紧密的结在质膜下，这种膜结迅速转变为小管，这些小管在富含肌动蛋白的皮层上辐射出去，这些小管在充盈阶段相互融合并与未成熟的膀胱融合，形成新的成熟膀胱膀胱。这些皮层事件在贴壁细胞腹侧表面的延时共焦图像中观察得最好，因为这种配置将大面积的质膜和下面富含肌动蛋白的皮层置于单个焦平面内。 CV 膜与富含肌动蛋白的皮质的紧密联系，以及 CV 小管沿着皮质的剧烈运动，表明 CV 膜募集某种类型的肌球蛋白。我们现在表明，盘基网柄菌 V 型肌球蛋白 myoJ 靶向于 CV 膜，并负责其与富含肌动蛋白的皮层的稳态关联。此外，我们发现，在水排出后，myo J 驱动塌陷的膀胱膜沿着皮层形成管状。最后，在 myoJ null 细胞中看到的 MTOC 周围 CV 膜的稳态积累迫使我们可视化细胞中部及其腹侧表面的 CV 膜动态。这些图像显示，从塌陷的膀胱发出的小管不仅在膜平面上的肌动蛋白上移动，而且沿着皮层和邻近细胞核的微管组织中心（MTOC）之间的微管双向移动。由此我们得出结论，myoJ 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功能。 稀（肌球蛋白 Va-）、灰白（Rab27a-）和铅色（亲黑素-）小鼠的毛色表型是相同的且非累加性，因为这些基因产物中任何一种的缺失都会完全消除黑素体与肌动蛋白细胞骨架的相互作用。我们已经开始表征稀释抑制子（dsu），这是一种稀释、灰白和铅的外源抑制子，我们的合作者最近通过回交和 bac 转基因拯救鉴定了它。 Dsu 编码一种约 22 kDa 的新型高电荷蛋白质。与 dsu 等位基因中存在的缺失突变一致，dsu 黑素细胞缺乏 dsu 蛋白 (dsup)。因此，dsup表达的丧失导致了救援。同样是纯合 dsu 的稀释黑素细胞显示黑素体在整个细胞质中显着扩散，这很可能解释了毛色的恢复。但这些细胞中的黑素体从不集中在树突尖，大概是因为这个过程严格依赖于肌球蛋白 Va。一致地，在稀释/dsu 黑素细胞中过度表达 GFP 标记的 dsup 会导致黑素体再次聚集在细胞中心。此外，这些细胞表现出 GFP 标记的 dsup 与黑素体几乎唯一的共定位。 dsup 的黑素体靶向需要在位于其 N 末端附近的六个半胱氨酸簇上添加多个棕榈酸酯，这表明 dsup 可能是专门的黑素体膜微域的组成部分。我们现在正在表征缺乏和过度表达 dsup 的黑素细胞中基于微管的黑素体运动的性质，以确定 dsup 是否通过影响微管上黑素体的正负末端定向运动之间的平衡来引起救援。