A Genetic Approach to the Study of Vesicle Transport in

几年前，我们发现小鼠自发突变稀释（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哺乳动物突变。稀释的小鼠产生正常量的色素，但是黑素体，即黑素细胞中含有色素的囊泡，不能有效地从细胞的核周区域运输到树突尖端。这会导致颜料释放不均匀和毛色稀释。随后，我们发现d编码一个基于肌动蛋白的分子马达MYO 5A。酵母双杂交研究表明，MYOVA作为具有基于微管的马达KIF 5 B的复合物的一部分发挥作用。这是第一个证据表明，一个整合的运动复合体可以在微管和肌动蛋白轨道上移动，我们现在正在通过产生黑素细胞中缺乏KIF 5 B的小鼠来测试这一假设。我们使用定位克隆来鉴定ashen（ash）和leaden（ln）的产物，这两种突变在表型学上被稀释。在随后的合作研究中，我们发现编码RAB 27 A的ash和编码RAB效应子的ln（称为Melanophilin（MLPH））蛋白在黑素体上形成复合物，作为MYOVA的受体发挥作用。这是第一个在哺乳动物囊泡上分子马达受体的分子描述。重要的是，这三种蛋白质在人类疾病中都会发生突变。Griscelli综合征是一种罕见的常染色体隐性遗传病，所有患者的皮肤和头发都显示色素减退。有些还显示不同严重程度的神经功能障碍，这与MYO 5A突变相关;其他人除了色素减退外还显示免疫功能障碍，这些表型与RAB 27 A突变相关。携带稀释的各种等位基因的小鼠，也可以显示神经异常，也可以显示免疫异常，现在是进一步研究这些人类疾病的极好模型。MLPH突变患者仅显示色素减退表型，这与在ln突变纯合子小鼠中未检测到其他表型的观察结果一致。在尚未提交发表的研究中，我们成功地定位克隆了稀释抑制基因（dsu），这是在哺乳动物中发现的第一个非连锁抑制基因突变。DSU是一种半显性抑制因子，其至少部分地挽救D、Ash和LN小鼠的毛色稀释。与我们的预期相反，dsu不编码马达蛋白，或可以调节马达蛋白并替代MYO 5A缺失的蛋白质（即转录因子）。相反，我们发现dsu编码的是一种没有明显功能基序的小蛋白。因此，DSU可能代表调节定向囊泡运输的新途径的创始成员。我们还发现了dsu、d、ash和ln在眼睛中的惊人作用，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（ru 2）和红宝石眼（ru），这是另外两种被dsu部分抑制的色素稀释突变。我们发现，由ru 2和ru编码的蛋白质形成蛋白质复合物，并分别在人类Hermansky-Pudlak综合征（HPS）5型和6型患者中发生突变。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，合作研究显示，由桑迪色素稀释突变编码的基因在HPS 7型患者中发生突变。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。

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.