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Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.

Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.

Important progress has been made during the past decade in the identification of molecular motors required in the distribution of early and late endosomes and the proper trafficking along the endocytic pathway. There is little direct evidence, however, that these motors drive movement of the endosomes. To evaluate the contributions of kinesin-1, dynein and kinesin-2 to the movement of early and late endosomes along microtubules, we made use of a cytosol-free motility assay using magnetically isolated early and late endosomes as well as biochemical analyses and live-cell imaging. By making use of specific antibodies, we confirmed that kinesin-1 and dynein move early endosomes and we found that kinesin-2 moves both early and late endosomes in the cell-free assay. Unexpectedly, dynein did not move late endosomes in the cell-free assay. We provide evidence from disruption of dynein function and latrunculin A treatment, suggesting that dynein regulates late endosome movement indirectly, possibly through a mechanism involving the actin cytoskeleton. These data provide new insights into the complex regulation of endosomes' motility and suggest that dynein is not the major motor required to move late endosomes toward the minus end of microtubules.

Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of membrane delivery required for optimal FcR-mediated phagocytosis.

Cystic fibrosis (CF), due to mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), exhibits a wide range of disease severity, even among deltaF508 homozygous patients, and the mechanisms of this variability have yet to be elucidated. In view of the close structural homology and possible functional overlap between CFTR and Multidrug Resistance-associated Proteins (MRPs), MRPs were investigated as potentially relevant factors in CF pathophysiology. MRP1-5 gene expression was analyzed in nasal respiratory epithelial cells from deltaF508 homozygous patients (n = 19) and control subjects (n = 20) using semiquantitative RT-PCR. Significantly lower MRP1 and MRP5 transcript levels were found in CF patients than in control subjects. MRP1 and MRP5 transcript levels were strongly correlated (r = 0.71). In CF patients, low MRP1 transcript levels were associated with more severe disease as assessed by the Shwachman score. A relation was also observed between MRP1 levels and presence of a cAMP-independent chloride conductive pathway, as determined by a halide-sensitive fluorescent assay. These results suggest that MRPs, especially MRP1, might play a role in CF phenotype and might therefore constitute a target for a novel pharmacotherapy of CF.

The aim of our study was to investigate the functional expression of P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) in 2 distinct glioma cells (GL15 and 8MG) from patients with glioblastoma multiforme. MDR1 gene and Pgp expression was not detected in either cell line by RT-PCR and Western blotting, respectively. In contrast, MRP1 was detected at both mRNA and protein level in both cell lines, with a higher expression in the 8MG cells that occur predominantly at the cell membrane. Three other MRPs (MRP3, MRP4 and MRP5) were detected by RT-PCR in both cell lines, whereas MRP2 was not expressed. In addition, MRP3 protein was also detected by immunocytochemistry in both GL15 and 8MG cell lines. Indomethacin and probenecid, 2 modulators of MRPs activity, increased the accumulation of vincristine and etoposide, 2 substrates of MRPs, by both cell lines. These modulators also decreased the efflux of vincristine from both cell lines with a more pronounced effect in 8MG cells. In conclusion, our results show functional expression of MRPs leading to a decrease in the intracellular vincristine and etoposide concentrations in human glioblastoma cell lines. Furthermore, our results that exhibit protein expression of MRP1 and MRP3 and gene expression of MRP4 and MRP5 in these 2 glioblastoma cell lines suggest new mechanisms that could lead to a MDR phenotype of tumour cells in patients with glioblastoma multiforme., (Copyright 2002 Wiley-Liss, Inc.)

The MRP (multidrug resistance-associated protein) transmembrane transporter, which actively transports a wide variety of lipophilic substrates out of cancer cells, has been suggested to play a major role in cell detoxification via efflux of glutathione conjugates. Because bronchial epithelial cells are constantly exposed to environmental pollutants, MRP might be a particularly important defense mechanism against xenobiotics. This study was therefore designed to investigate MRP localization by immunohistochemistry in bronchial epithelial cells collected by scraping from surgical specimens. In parallel, MRP mRNA was detected by reverse transcriptase chain reaction (rt-PCR) in bronchial cell lysates. However, the pattern of protein expression differed markedly according to cell type. In ciliated epithelial cells, immunostaining was restricted to the basolateral surface, without any labeling at the apical surface, which is at variance with the localization of CFTR and MDR1 proteins, other members of the same family of transporters. In basal cells, MRP was present over the entire circumference of the plasma membrane. Basal cells were identified by their morphology and specifically after incubation with an anticytokeratin 17 monoclonal antibody. In conclusion, the different patterns of localization suggest specific roles for MRP in basal and ciliated cells.

The main oxidizing free radicals (FR) are oxygen-derived metabolites (superoxide anion O2-, hydrogen peroxide H2O2, hydroxyl radical OH (zero)), hypochloric acid, chloramines, nitrogen dioxide, ozone and lipid peroxides. They are produced continually by living organisms, either in the intracellular compartment by the mitochondrial respiratory chain and mixed function oxidase system, or in the extracellular compartment, especially by phagocytes. The body possesses complex protective antioxidant systems against this potentially toxic production, such as dismutase superoxides, catalase, the glutathione enzyme system, metallic ion sequestration, enzymes degrading proteins injured by FR, metabolising hydroperoxides, and repairing DNA, and vitamins E, C, P, and betacarotene. A physiological steady-state is established under normal conditions between the production of oxidants and their neutralization by antioxidants. Oxidative lesions result from a disturbance of the oxidant-antioxidant balance. Oxygen-derived metabolites act on polyunsaturated cell membrane lipids, induce genetic alterations, and oxidize sulfhydryl groups of proteins, thereby modifying their functions. FR are involved in major physiological mechanisms such as phagocytosis, the inflammatory reaction, and the reperfusion ischaemia phenomenon observed during organ storage. The therapeutic use of enzymatic (SOD, catalase, GSH) and nonenzymatic antioxidants (vitamins E, N-acetylcysteine, allopurinol and oxypurinol) has yet to be evaluated. The current state of our knowledge indicates the extreme complexity of these systems and calls for caution in the therapeutic use of antioxidant substances.

After a brief review of oxygen-derived free radicals formation, cellular antioxidant equipment, and in vivo oxidant lung injury, the model of type II pneumocytes in primary culture is described with its characteristics and limitations. This model has been used to study either the in vivo or the in vitro effects of free radicals: the resistance of type II cells isolated from animals exposed to oxidants may be explained by their higher level of anti-oxidant enzymes. In contrast, type II cells isolated from control animals do not develop a resistance to the toxic effects on an in vitro exposure to free radicals. This discrepancy might be the result of a possible interaction of alveolar macrophages or fibroblasts with type II cells, which remains to be explored.