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.

微管加末端跟踪蛋白或 +尖端已与微管动力学，细胞极性，纺锤体定位和细胞器的控制有关：在依赖Dynein依赖性细胞器运动之前的微管相互作用。我们已经表明，小鼠黑素细胞中黑素体的分布是由远距离，双向，微管依赖性运动以及局部肌球蛋白VA依赖性运动驱动的。我们还表明，肌球蛋白VA是通过含有黑色素体膜上的Rab27a和黑色素蛋白上的Rab27a的受体复合物募集到黑色素体表面的，将肌球蛋白VA与RAB27A联系起来。现在，我们表明，GFP标签的黑色素蛋白还表现出微管和最终跟踪行为。此外，GFP标记的肌球蛋白VA还可以浏览微管加上结束，并以黑色素蛋白依赖性方式进行。最后， +尖端EB1的细胞水平的变化以及下拉测定法认为，黑素蛋白通过在EB1上搭便车而间接浮出水面。这些结果表明，脊椎动物细胞以肌球蛋白VA-Melanophilin-Eb1复合物的形式保留了微管加的末端和F-肌动蛋白之间的连接，其中包含MyO2p（一种V型V肌蛋白），BIM1P（A EB1同源性）和KAR9P，链接MyoO2P与BiM1p的KAR9P。鉴于黑色素蛋白在将含拉乳胶的囊泡货物耦合到肌球蛋白VA中所记录的作用，我们建议在微管加末端的黑色素蛋白和肌球蛋白VA的积累可能有助于聚焦并促进黑色素体从微管中转移到该位置的Actin。 收缩液泡（CV）复合物是一种专门的细胞内膜室，用作原生动物中的osmoregulation细胞器。在dictyostelium中，该室由小管和蓄水池或膀胱的互连网络组成。这些膜积聚了多余的水（例如雨水），通过渗透质子并最有可能将碳酸氢盐泵入渗透。所得的离子梯度将多余的水从细胞质中抽出并进入管腔。产生的肿胀的膀胱通过质膜中的瞬时融合孔从细胞中排出。组成系统的小管和膀胱是高度动态的，非常多形，可快速互连，并且在水驱动过程中不会与内体/溶酶体膜系统或质膜混合。先前的研究所产生的是对CV膜周期的工作定义，在该定义中，肿胀，成熟的膀胱接触质膜中的融合孔，从细胞中排出水，倒塌的膀胱膜折叠成紧密的结中，紧密地构成了质膜，跨越了质膜的旋转，该旋转均匀地变成了束时，该旋转构成了束时，该支着膜，呈现出偏变的态度，该旋转均匀地构成了束缚，该旋转均匀地构成了束时，该旋转均匀地挤满了束时，该乳剂呈现出繁殖的繁殖，该旋转是在束缚的范围。这些小管相互融合，并在填充阶段与未成熟的膀胱融合，以创建新的成熟膀胱。这些皮质事件在粘附细胞的腹侧表面的延时共聚焦图像中被认为是最佳的，因为这种构型将质膜的大面积和次级肌动蛋白富含皮质的大面积放在单个焦平面内。 CV膜与富含肌动蛋白的皮质的密切关联，以及沿皮质的CV小管的戏剧性运动性，导致CV膜募集了某种类型的肌球蛋白。现在，我们表明，Dictyostelium型V肌球蛋白MyoJ针对CV膜，并负责其与富含肌动蛋白富含皮质的稳定状态关联。此外，我们表明Myo J在排水后沿皮质沿皮质塌陷的膀胱膜的管道驱动。最后，在MyoJ无效细胞中看到的MTOC周围的CV膜的稳态积累迫使我们可视化细胞中间的CV膜动力学以及沿其腹侧表面。这些图像表明，塌陷的膀胱发出的小管不仅在膜平面上在肌动蛋白上移动，而且在毗邻核的微管和微管组织中心（MTOC）之间沿着微管进行双向双向移动。由此我们得出的结论是，Myoj与Plus和Minus Dient指导的微管电动机合作，以驱动Dictyostelium中简历复合物的适当分布和功能。 稀释（肌球蛋白VA-），灰（Rab27a-）和铅（黑色素蛋白 - ）小鼠的外套颜色表型是相同且非辅助的，因为这些基因产物中的任何一种完全消除了黑色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剂（DSU），这是一种稀释，灰烬和铅的外部抑制器，我们的合作者最近通过Back Crossing和BAC Transgene Rescue确定了这些抑制剂。 DSU编码了一种〜22 kDa的新型，高电荷的蛋白质。与DSU等位基因中存在的缺失突变一致，DSU黑色素细胞没有DSU蛋白（DSUP）。因此，DSUP的表达丧失导致救援。也是纯合DSU的稀黑色素细胞在整个细胞质中显示出明显的黑素体传播，这很可能解释了涂层颜色的恢复。但是这些细胞中的黑素体从未集中在树突状尖端上，大概是因为该过程严格取决于肌球蛋白VA依赖性。一致地，在稀/DSU黑色素细胞中GFP标记的DSUP的过表达会导致黑素体再次聚集在细胞中心。此外，这些细胞几乎显示了GFP标记的DSUP与黑色素体的独家共定位。 DSUP的黑色素体靶向需要在其N末端附近的六个半胱氨酸的簇上添加多个棕榈酸盐，这表明DSUP可能是专门的黑色素体膜微结构域的组成部分。现在，我们正在表征缺乏和表达DSUP的黑色素细胞中基于微管的黑色素运动体运动的性质，以确定DSUP是否通过在微管上实现黑色素体和减去终端导向运动之间的平衡来确定救援。