A Genetic Approach to the Study of Vesicle Transport

研究囊泡运输的遗传学方法

• 批准号: 6951681
• 负责人: NANCY JENKINS COPELAND
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6951681
• 关键词: actins; albinism; alleles; animal genetic material tag; animal population genetics; autosomal recessive trait; biological transport; disease /disorder model; genetic disorder; intracellular transport; laboratory mouse; melanosomes; microtubules; molecular cloning; phenotype; pigmentation; pigments; platelet disorder; protein structure function; suppressor mutations; vesicle /vacuole; yeast two hybrid system

Several years ago we showed that the spontaneous mouse mutation dilute (d) was caused by the integration of a retrovirus into the mouse genome. This observation was very exciting because it was the first demonstration of a retrovirus-induced mutation in mammals. dilute mice make normal amounts of pigment but the melanosomes, the pigment-containing vesicles in the melanocyte, are inefficiently transported from the perinuclear region of cell to the dendritic tips. This results in an uneven release of pigment and a dilution of coat color. Subsequently, we showed that d encodes an actin-based molecular motor, MYO5A. Yeast two hybrid studies suggested that MYOVA functions as part of a complex with a microtubule-based motor, KIF5B. This was the first evidence for an integrated motor complex that can move on both microtubule and actin tracks and we are now testing this hypothesis by generating mice lacking KIF5B in melanocytes. We used positional cloning to identify the products of ashen (ash) and leaden (ln), two mutations that phenocopy dilute. In subsequent collaborative studies we showed that the ash, which encodes RAB27A, and the ln, which encodes a RAB effector designated Melanophilin (MLPH), proteins form a complex on the melanosome that functions as the receptor for MYOVA. This was the first molecular description of a receptor for a molecular motor on a vesicle in mammals. Importantly, all three proteins are mutated in human disease. All patients with Griscelli Syndrome, a rare autosomal recessive disorder, display hypopigmentation of the skin and hair. Some also show neurological impairment of varying severity, which correlates with mutations in MYO5A; others show immune dysfunction in addition to hypopigmentation and these phenotypes correlate with mutations in RAB27A. Mice carrying various alleles of dilute, which can also display neurological abnormalities, and of ashen, which also display immune abnormalities, are now excellent models for the further study of these human diseases. Patients with mutations in MLPH display only a hypopigmentation phenotype, consistent with the observation that no other phenotypes are detected in mice homozygous for the ln mutation. In studies that have not yet been submitted for publication we succeeded in positionally cloning dilute suppressor (dsu), the first unlinked suppressor mutation to be identified in mammals. dsu is a semidominant suppressor that rescues, at least partially, the coat color dilution of d, ash, and ln mice. Contrary to our expectations, dsu does not encode a motor protein, or a protein (i.e. a transcription factor) that could regulate a motor protein and substitute for the loss of MYO5A. Instead, we found that dsu encodes a small protein with no striking functional motifs. dsu may therefore represent the founding member of a new pathway that regulates directional vesicle transport. We also uncovered surprising effects of dsu, d, ash, and ln in the eye, a subject of future studies. In collaborative studies, we positionally cloned ruby eye-2 (ru2) and ruby-eye (ru), two other pigment diluting mutations that are partially suppressed by dsu. We showed that the proteins encoded by ru2 and ru form a protein complex and are mutated in human Hermansky-Pudlak syndrome (HPS) type 5 and 6 patients, respectively. HPS is a multigenic disorder that affects melanocytes, platelets, and lysosomes, organelles that share a common biosynthetic pathway. Very recently, collaborative studies revealed that the gene encoded by the sandy pigment diluting mutation is mutated in HPS type 7 patients. Again, mice carrying these various diluting mutations are very important models for further studying human disease.

几年前，我们证明了自发的小鼠突变稀释度(D)是由逆转录病毒整合到小鼠基因组中引起的。这一观察结果非常令人兴奋，因为这是第一次证明逆转录病毒诱导的哺乳动物突变。稀释的小鼠产生正常数量的色素，但黑素小体--黑素细胞中含有色素的囊泡--从细胞的核周区域低效率地运输到树突尖端。这会导致颜料的不均匀释放和毛色的淡化。随后，我们发现d编码了一个基于肌动蛋白的分子马达MYO5A。酵母双杂交研究表明，MYOVA作为具有微管马达KIF5B的复合体的一部分发挥作用。这是整合的运动复合体可以在微管和肌动蛋白轨迹上移动的第一个证据，我们现在正在通过产生黑素细胞中缺乏KIF5B的小鼠来检验这一假设。我们使用位置克隆来鉴定灰烬(Ash)和铅(Ln)的产物，这两个突变会稀释表型。在随后的合作研究中，我们发现编码RAB27A的ASH和编码RAB效应器的Ln(MLPH)蛋白质在黑素小体上形成一个复合体，作为MYOVA的受体。这是首次对哺乳动物囊泡上的分子马达受体进行分子描述。重要的是，在人类疾病中，这三种蛋白质都发生了突变。Griscelli综合征是一种罕见的常染色体隐性遗传病，所有患者都表现出皮肤和头发色素沉着减少。有些还表现出不同程度的神经损害，这与MYO5A突变有关；另一些表现出免疫功能障碍，除了色素减少，这些表型与RAB27A突变有关。携带各种稀释性和灰白色等位基因的小鼠现在是进一步研究这些人类疾病的极佳模型。稀释性和灰白色也会显示出神经异常。具有MLPH突变的患者仅表现为色素减退表型，这与观察到在纯合Ln突变的小鼠中没有检测到其他表型一致。 在尚未提交发表的研究中，我们成功地定位克隆了稀释型抑制子(DSU)，这是在哺乳动物中发现的第一个非连锁抑制子突变。Dsu是一种半显性抑制物，至少部分地挽救了d、ash和n小鼠的毛色淡化。与我们的预期相反，DSU不编码马达蛋白，也不编码可以调节马达蛋白并替代MYO5A缺失的蛋白质(即转录因子)。相反，我们发现DSU编码的是一个小蛋白，没有明显的功能基序。因此，DSU可能代表了一条调控囊泡定向运输的新途径的创始成员。我们还发现了dsu、d、ash和ln在眼睛中的惊人影响，这是未来研究的主题。 在合作研究中，我们定位克隆了红宝石眼-2(Ru2)和红宝石眼(Ru)，这是另外两个被DSU部分抑制的色素稀释突变。我们发现RU2和Ru编码的蛋白质形成蛋白质复合体，在人类Hermansky-Pudlak综合征(HPS)5型和6型患者中分别发生突变。HPS是一种多基因疾病，影响黑素细胞、血小板和溶酶体，这些细胞器共享共同的生物合成途径。最近，合作研究发现，由沙色色素稀释突变编码的基因在HPS 7型患者中发生了突变。同样，携带这些不同稀释突变的小鼠是进一步研究人类疾病的非常重要的模型。