Lanfredi-Rangel A, Attias M, Reiner D S, Gillin F D, De Souza W
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Bloco G-SS, Av Brig Trompowsky, s/no, CEP 21949-900, Rio de Janeiro, RJ, Brazil.
J Struct Biol. 2003 Aug;143(2):153-63. doi: 10.1016/s1047-8477(03)00123-0.
Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.
细胞外囊壁的合成、运输和组装是蓝氏贾第鞭毛虫包囊化的标志。我们对其生化途径及其调控已经了解很多。然而，从细胞生物学的角度来看，将囊壁蛋白运输到细胞周边的包囊化特异性囊泡（ESV）的生物发生过程却知之甚少。因此，我们采用了多种互补的超微结构方法来验证这一假说，即ESV的形成利用了一种新型的受调控分泌途径。我们通过对薄切片、冷冻断裂复制品进行电子显微镜观察，以及对固定用于内质网（ER）标记物葡萄糖6-磷酸酶细胞化学定位的细胞连续切片进行三维重建，分析了体外不同包囊化阶段的寄生虫。我们还使用了体视学方法来确定内质网、裂隙、ESV和囊壁所占的面积。综合我们的动力学数据表明，一些内质网池首先扩张形成裂隙，然后扩大为ESV。活的未包囊化和早期包囊化滋养体在两个细胞核周围的周边区域用C(6)-NBD-神经酰胺进行标记。在18 - 21小时时，一些ESV向外迁移经常导致包囊化滋养体周边出现突起。使用酸性区室标记物吖啶橙证实了在ESV和细胞的质膜之间存在溶酶体样周边囊泡。我们的数据表明，蓝氏贾第鞭毛虫具有一种新型分泌途径，其中内质网和高尔基体的某些功能在空间和时间上共定位。这些研究将增进我们对早期分化真核生物中用于组装原始细胞外基质的受调控分泌途径进化出现的理解。
