Endocytic Trafficking and Human Diseases

粘磷脂构成了一个与瞬时受体电位超家族同源的内体阳离子通道家族。在哺乳动物中，粘蛋白家族包括三个成员，即粘蛋白-1、-2和-3(MCOLN1-3)。MCOLN1是该家族中特征最好的成员，因为这种蛋白的突变与一种称为IV型粘脂沉积症(MLIV)的人类疾病有关。MLIV是一种常染色体隐性遗传病，其特征是智力和精神运动发育迟缓、肌肉张力降低或低眼压、失弛缓症以及包括角膜混浊、视网膜变性、对光敏感和斜视在内的视觉问题。对MLIV患者成纤维细胞的电子显微镜分析显示，存在大量的空泡结构，这些结构积聚了粘多糖和脂类，形成了具有特征的多同心片层。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，表明溶酶体功能普遍受损。 我们以前的研究表明，MCOLN1介导了晚期内小体和溶酶体的瞬时、调节和局部的钙外流。腔内钙的释放在几个转运事件中起着关键作用，包括溶酶体与自噬小体、晚期内体和质膜的融合。与这一观点一致，我们发现MCOLN1是沿着内体途径进行适当的膜运输和维持离子稳态所必需的。 虽然原代细胞和细胞系的使用为了解MCOLN1的功能提供了关键信息，但进一步验证这些体外研究和体内观察将是非常重要的。出于这个原因，我们目前正在研究斑马鱼MCOLN1基因敲除的世代。使用斑马鱼作为我们正在进行的研究的潜在有用的模型是基于一系列特征，包括它们很容易在狭小的实验室空间中大量维护，它们的繁殖时间相对较短，并且它们在胚胎学分析中呈现出独特的优势。具体地说，我们正在通过注射特定的吗啡来诱导MCOLN1的急性耗竭，以及通过使用锌指核酸酶来产生稳定的基因工程MCOLN1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.