Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.

Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells.

Cystogenesis associated with autosomal dominant polycystic kidney disease (ADPKD) is characterized by perturbations in the polarized phenotype and function of cyst-lining epithelial cells. The polycystins, the protein products of the genes mutated in the majority of ADPKD cases, have been described recently, but the pathological mechanism by which causal mutations result in the mislocalization of cell membrane proteins has remained unclear. This report documents the dissociation from the ADPKD cell basolateral membrane of three molecules essential for spatial organization and exocytosis. The adherens junction protein E-cadherin, the subcellular disposition of which governs intercellular and intracellular architecture, was discovered sequestered in an internal ADPKD cell compartment. At the same time, sec6 and sec8, components of a complex critical for basolateral cargo delivery normally arrayed at the apico-lateral apex, were depleted from the ADPKD cell plasma membrane. An analysis of membrane transport revealed that basolateral trafficking of proteins and lipids was impaired as a result of delayed cargo exit from the ADPKD cell Golgi apparatus. Apical transport proceeded normally. Taken together with recent documentation of an association between polycystin-1 and E-cadherin (Huan and van Adelsberg 1999), the data suggest that causal mutations disrupt E-cadherin-dependent cytoarchitecture, adversely affecting protein assemblies crucial for basolateral trafficking.

ADPKD: a human disease altering Golgi function and basolateral exocytosis in renal epithelia.

Epithelial cells explanted from autosomal dominant polycystic kidney disease (ADPKD) tissue exhibit impaired exocytosis, specifically between the Golgi and basolateral membrane (Charron A, Nakamura B, Bacallo R, Wandinger-Ness A. Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells. J Cell Biol 2000; 148: 111-124.). Here the defect is shown to result in the accumulation of the basolateral transport marker vesicular stomatitis virus (VSV) G protein in the Golgi complex. Golgi complex morphology is consequently altered in the disease cells, evident in the noticeable fenestration and dilation of the cisternae. Further detailed microscopic evaluation of normal kidney and ADPKD cells revealed that ineffective basolateral exocytosis correlated with modulations in the localization of select post-Golgi transport effectors. The cytosolic coat proteins p200/myosin II and caveolin exhibited enhanced association with the cytoskeleton or the Golgi of the disease cells, respectively. Most cytoskeletal components with known roles in vesicle translocation or formation were normally arrayed with the exception of Golgi beta-spectrin, which was less prevalent on vesicles. The rab8 GTPase, important for basolateral vesicle targeting, was redistributed from the perinuclear Golgi region to disperse vesicles in ADPKD cells. At the basolateral membrane of ADPKD cells, there was a notable loss of the exocyst components sec6/sec8 and an unidentified syntaxin. It is postulated that dysregulated basolateral transport effector function precipitates the disruption of basolateral exocytosis and dilation of the ADPKD cell Golgi as basolateral cargo accumulates within the cisternae.

Cablin: a novel protein of the capillary basal lamina.

The microvascular wall is remarkably simple, consisting only of the endothelial lining, subjacent basal lamina, and underlying periendothelial cells. This study describes the characterization of a novel microvascular protein. This 80,000-molecular weight protein was predominantly associated with electron-lucent amorphous material in capillary basal laminae and therefore termed cablin (protein of the capillary basal lamina). Consistent with its immunolocalization to the microvasculature, cablin was synthesized and secreted by cultured endothelial cells and vascular smooth muscle cells. Furthermore, cablin expression was induced during neovascularization. The predicted amino acid sequence of cablin revealed a prevalence of polar amino acids. Accounting for the low yet significant homology to several alpha-helical proteins, these residues were best accommodated by secondary structure predictions that aligned the molecule into two large alpha-helical domains. The presence of the integrin-binding RGD tripeptide and a putative elastin-binding sequence suggest that this rodlike molecule is suited to cross-link cells and matrix constituents. In this capacity it could contribute to the mechanical strength or the angiogenic potential of the microvasculature.

Investigation of myelin/oligodendrocyte glycoprotein membrane topology.

Myelin/oligodendrocyte glycoprotein (MOG) is a CNS-specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain-specific antisera to ascertain the localization of these hydrophobic domains. As expected, MOG's glycosylated N-terminal Ig-like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG1-125 antiserum. In contrast, both MOG154-169 and MOG198-218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N-terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C-terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane-associated) would be unique among Ig-like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.