Endocytic Trafficking and Human Diseases

粘脂构成了一个家族的内体阳离子通道与瞬时受体电位超家族同源。在哺乳动物中，粘脂家族包括三个成员，粘脂-1、粘脂-2和粘脂-3（MCOLN 1 -3）。MCOLN 1是该家族中最具特征的成员，因为这种蛋白质的突变与称为IV型粘脂沉积症（MLIV）的人类疾病相关。MLIV是一种常染色体隐性遗传疾病，其特征在于智力和精神发育迟缓、肌张力降低或张力减退、无氯血症和视觉问题，包括角膜混浊、视网膜变性、对光敏感和斜视。通过电子显微镜分析MLIV患者的成纤维细胞，发现存在扩大的空泡结构，积累粘多糖和脂质，形成特征性的多中心层。这些扩大的空泡不仅存在于成纤维细胞中，而且存在于MLIV患者的每个组织和器官中，表明溶酶体功能的普遍损害。 我们以前的研究表明，MCOLN 1介导的瞬时，调节，并从晚期内体和溶酶体局部钙外流。管腔钙的释放在几种运输事件中起关键作用，包括溶酶体与自噬体、晚期内体和质膜的融合。与这一观点一致，我们发现MCOLN 1是沿着内体途径沿着进行适当的膜运输和维持离子稳态所必需的。 尽管原代细胞和细胞系的使用为理解MCOLN 1的功能提供了关键信息，但通过体内观察进一步验证这些体外研究将非常重要。出于这个原因，我们目前正在斑马鱼中产生MCOLN 1敲除。使用斑马鱼作为我们正在进行的研究的一个潜在的有用的模型是基于一些功能，包括它们很容易保持在一个小的实验室空间中大量，它们的世代时间相对较短，他们提出了独特的优势，胚胎学分析。具体来说，我们正在研究通过注射特定的morpholionos诱导急性MCOLN 1耗竭，以及通过使用锌指核酸酶产生稳定的基因工程MCOLN 1敲除斑马鱼。

Mucolipins constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3). MCOLN1 is the best-characterized member of the family due to the fact that mutations in this protein are associated with a human disease known as mucolipidosis type IV (MLIV). MLIV is an autosomal recessive disease characterized by mental and psychomotor retardation, diminished muscle tone or hypotonia, achlorhydria, and visual problems including corneal clouding, retinal degeneration, sensitivity to light, and strabismus. Analysis of fibroblasts from MLIV patients by electron microscopy revealed the presence of enlarged vacuolar structures that accumulate mucopolysaccharides and lipids forming characteristic multiconcentric lamellae. These enlarged vacuoles are present not only in fibroblasts but in every tissue and organ of MLIV patients, suggesting a general impairment of the lysosomal function. Our previous studies revealed that MCOLN1 mediates transient, regulated, and localized calcium efflux from late endosomes and lysosomes. Release of luminal calcium plays a pivotal role in several trafficking events, including fusion of lysosomes with autophagosomes, late endosomes, and plasma membrane. In agreement with this idea we found that MCOLN1 is required for proper membrane trafficking along the endosomal pathway and maintenance of ion homeostasis. Althoug the use of primary cells and cell lines has provided crucial information for understanding the function of MCOLN1, it will be of great importance to further validate these studies done in vitro with in vivo observations. For this reason, we are currently working in the generation of MCOLN1 knockouts in zebrafish. The use of zebrafish as a potentially useful model for our ongoing studies is based on a number of features, including the fact that they are easy to maintain in large numbers in a small laboratory space, their generation time is relatively short, and they present unique advantages for embryological analysis. Specifically we are working in the induction of acute MCOLN1 depletion by injection with specific morpholionos as well as in the generation of stable genetically engineered MCOLN1-knockout zebrafish by the use of Zinc Finger Nucleases.