Developmental control of organelle transport

描述（由申请人提供）：本提案的长期目标是了解允许细胞器运输与发展相结合的调节机制。沿着微管的马达驱动运输在细胞中扮演着许多重要的角色，然而细胞控制其特异性、时间和目的地的机制仍然是一个谜。一个新开发的模型系统，果蝇胚胎中的脂滴运动，提供了一个利用遗传、生物物理和分子技术的跨学科方法来解决细胞生物学中这个长期存在的问题的机会。1)提出了Halo蛋白作为一种新型的反式作用调节因子，如何介导液滴运输中的发育转变的实验。将监测Halo的时间表达和细胞内定位，以测试Halo是否是决定运输时间和方向的信号，以及Halo是否与液滴上的马达物理关联。结构-功能分析将用于生成Halo的生化活性模型。利用光学镊子对野生型和突变型胚胎进行纳米级跟踪和失速力测量，将在单细胞器水平上确定Halo控制的运动物理参数。2)为了确定运输方向性的其他决定因素，将分离改变净液滴运输的新突变。由这些突变定义的基因将被分子克隆，它们与液滴运输机制的已知组分的物理和功能相互作用将被建立。对其他运输途径也很重要的基因将优先分析，以确定细胞器运动的关键一般调节因子。3)为了研究转运的细胞器特异性是如何建立的，我们将比较细胞质动力蛋白对几种胚胎货物的靶向性。特别是，我们将验证细胞质动力蛋白中间链的不同同工异构体将马达引入卵黄囊泡、脂滴和线粒体的假设。了解微管运动是如何被控制的对人类健康具有广泛的意义，因为异常的运动功能与从出生缺陷到癌症、精神分裂症和阿尔茨海默病等疾病有关。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to understand the regulatory mechanisms that allow organelle transport to be integrated with development. Motor-driven transport along microtubules plays many essential cellular roles, yet the mechanisms with which cells control its specificity, timing and destination remain a mystery. A newly developed model system, lipid-droplet motion in Drosophila embryos, provides the opportunity to attack this long-standing problem in cell biology with an interdisciplinary approach utilizing genetic, biophysical, and molecular techniques. 1) Experiments are proposed to establish how the Halo protein, a novel trans-acting regulator, mediates developmental transitions in droplet transport. The temporal expression and intracellular localization of Halo will be monitored to test whether Halo is the signal that determines timing and directionality of transport and whether Halo physically associates with motors on droplets. A structure-function analysis will be employed to generate models about Halo's biochemical activity. Nanometer-scale tracking and stall-force measurements with optical tweezers in wild-type and mutant embryos will determine at the single-organelle level which physical parameters of motion Halo controls. 2) To identify additional determinants of transport directionality, new mutations altering net droplet transport will be isolated. The genes defined by these mutations will be molecularly cloned, and their physical and functional interactions with known components of the droplet transport machinery will be established. Genes also important for other transport pathways will be preferentially analyzed to define key general regulators of organelle motility. 3) To investigate how organelle-specificity of transport is established, the targeting of cytoplasmic dynein to several embryonic cargoes will be compared. In particular, the hypothesis will be tested that different isoforms of the intermediate chain of cytoplasmic dynein recruit the motor to yolk vesicles, lipid droplets, and mitochondria. Understanding how microtubule motors are controlled has broad significance for human health since aberrant motor function has been linked to diseases ranging from birth defects to cancer, schizophrenia and Alzheimer's disease.