Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors.
(65/1021)
Members of the p24 family of type I transmembrane proteins are highly abundant in transport vesicles and are thought to be involved in selective protein transport between the endoplasmic reticulum and the Golgi complex. The p24 proteins have been grouped into four subfamilies (alpha, beta, gamma, and delta) and appear to assemble into tetrameric complexes that contain only one representative from each subfamily. Here we molecularly dissected the p24 family in a single cell type, namely in the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis. The biosynthetic activity of these cells for production of their major cargo protein proopiomelanocortin (POMC) can be physiologically manipulated via the process of background adaptation (similar30-fold induction, with highly active cells in black toads and virtually inactive cells in white animals). Extensive cDNA library screening revealed the identity of six p24 proteins expressed in the Xenopus melanotrope cells, namely one member of the p24alpha (alpha(3)), one of the p24beta (beta(1)), two of the p24gamma (gamma(2), gamma(3)) and two of the p24delta (delta(1), delta(2)) subfamily. Two other Xenopus p24 proteins, Xp24alpha(2) and -gamma(1), were not expressed in the melanotrope cells, pointing to cell-type specific p24 expression. Of the six melanotrope p24 proteins, the expression of four (Xp24alpha(3), -beta(1), -gamma(3) and -delta(2)) was 20- to 30-fold induced in active versus inactive melanotropes, whereas that of the other two members (Xp24gamma(2) and -delta(1)) had not or only slightly increased. The four proteins were induced only in the intermediate melanotrope cells and not in the anterior pituitary cells, and displayed similar overall tissue distributions that differed from those of Xp24gamma(1), -gamma(2) and -delta(1). Together, our results reveal that p24 expression can be cell-type specific and selectively induced, and suggest that in Xenopus melanotrope cells an alpha(3)/beta(1)/gamma(3)/delta(2) p24 complex is involved in POMC transport through the early stages of the secretory pathway. (+info)
Photochemical disruption of endocytic vesicles before delivery of drugs: a new strategy for cancer therapy.
(66/1021)
The development of methods for specific delivery of drugs is an important issue for many cancer therapy approaches. Most of macromolecular drugs are taken into the cell through endocytosis and, being unable to escape from endocytic vesicles, eventually are degraded there, which hinders their therapeutic usefulness. We have developed a method, called photochemical internalization, based on light-induced photochemical reactions, disrupting endocytic vesicles specifically within illuminated sites e.g. tumours. Here we present a new drug delivery concept based on photochemical internalization-principle -- photochemical disruption of endocytic vesicles before delivery of macromolecules, leading to an instant endosomal release instead of detrimental stay of the molecules in endocytic vesicles. Previously we have shown that illumination applied after the treatment with macromolecules substantially improved their biological effect both in vitro and in vivo. Here we demonstrate that exposure to light before delivery of protein toxin gelonin improves gelonin effect in vitro much more than light after. However, in vitro transfection with reporter genes delivered by non-viral and adenoviral vectors is increased more than 10- and six-fold, respectively, by both photochemical internalization strategies. The possible cellular mechanisms involved, and the potential of this new method for practical application of photochemical internalization concept in cancer therapy are discussed. (+info)
Imaging of procollagen transport reveals COPI-dependent cargo sorting during ER-to-Golgi transport in mammalian cells.
(67/1021)
We have examined the ER-to-Golgi transport of procollagen, which, when assembled in the lumen of the ER, is thought to be physically too large to fit in classically described 60-80 nm COPI- and COPII-coated transport vesicles. We found that procollagen exits the ER via COPII- coated ER exit sites and is transported to the Golgi along microtubules in defined transport complexes. These procollagen-containing transport complexes are, however, distinct from those containing other cargo proteins like ERGIC-53 and ts-045-G. Furthermore, they do not label for the COPI coat complex in contrast to those containing ts-045-G. Inhibition of COPII or COPI function before addition of ascorbate, which is required for the folding of procollagen, inhibits export of procollagen from the ER. Inactivation of COPI coat function after addition of ascorbate results in the localisation of procollagen to transport complexes that now also contain ERGIC-53 and are inhibited in their transport to the Golgi complex. These data reveal the existence of an early COPI-dependent, pre-Golgi cargo sorting step in mammalian cells. (+info)
Optimal transfection with the HK polymer depends on its degree of branching and the pH of endocytic vesicles.
(68/1021)
We have recently reported that liposomes in combination with histidine (HK)-containing polymers enhanced the expression of luciferase in transfected cells. In transformed or malignant cell lines, branched HK polymers (combined with liposome carriers) were significantly more effective than the linear HK polymer in stimulating gene expression. In the current study, we found that the linear HK polymer enhanced gene expression in primary cell lines more effectively than the branched polymers. The differences in the optimal carrier (linear versus branched) were not due to initial cellular uptake, size of the complexes or level of gene expression. There was, however, a strong association between the optimal type of HK polymer and the pH of endocytic vesicles (P = 0.0058). By altering the percentage of histidines carrying a positive charge, the endosomal pH of a cell may determine the amount of DNA released from the linear or branched HK polymer. In the two cell lines in which the linear HK was the optimal polymer, the endocytic vesicles were strongly acidic with a pH of <5.0. Conversely, in the four cell lines in which the branched polymers were optimal transfection agents, the pH of endocytic vesicles was >6.0. Furthermore, binding data support the relationship between DNA release from the optimal HK polymer and endosomal pH. The interplay between optimal HK polymers and the endosomal pH may lead to improved gene-delivery polymers tailored to a particular cell. (+info)
Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress.
(69/1021)
We studied the effect of microtubule-associated tau protein on trafficking of vesicles and organelles in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells. Tau inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites. Loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration. In particular, tau inhibits transport of amyloid precursor protein (APP) into axons and dendrites, causing its accumulation in the cell body. APP tagged with yellow fluorescent protein and transfected by adenovirus associates with vesicles moving rapidly forward in the axon (approximately 80%) and slowly back (approximately 20%). Both movements are strongly inhibited by cotransfection with fluorescently tagged tau (cyan fluorescent protein-tau) as seen by two-color confocal microscopy. The data suggests a linkage between tau and APP trafficking, which may be significant in Alzheimer's disease. (+info)
TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex.
(70/1021)
We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes. (+info)
Separation of micelles and vesicles within lumenal aspirates from healthy humans: solubilization of cholesterol after a meal.
(71/1021)
Understanding the physico-chemical relationship of lumenal lipids to one another is critical when elucidating the mechanism of components known to impact cholesterol absorption. Presently, there are no studies that describe the proportion of cholesterol carried as micelles or vesicles within human lumenal contents. Part of the reason for the scarceness of data is because of the lack of appropriate methodology required for reproducible sample collection and analysis. Thus, the object of the present studies was to develop a method to measure the amount of cholesterol carried as micelles or vesicles in human lumenal samples. The method includes the collection of lumenal samples from the ligament of Trietz through a Fredrick Miller tube, separation of the aqueous subphase from the nondigested lipids, separation of micelles and vesicles on Sepharose 4B columns within 48 h of collection using elution buffers consisting of the intermicellar bile acid composition, and finally quantitation of cholesterol eluted off of the columns. The distribution of cholesterol between micelles and vesicles obtained under different concentrations of bile acids and various lipids was comparable to results obtained from phase diagrams using the lumenal molar percentages of lipids obtained from the same samples. (+info)
Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.
(72/1021)
Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner. (+info)