Palladin promotes invasion of pancreatic cancer cells by enhancing invadopodia formation in cancer-associated fibroblasts.

The stromal compartment surrounding epithelial-derived pancreatic tumors is thought to have a key role in the aggressive phenotype of this malignancy. Emerging evidence suggests that cancer-associated fibroblasts (CAFs), the most abundant cells in the stroma of pancreatic tumors, contribute to the tumor's invasion, metastasis and resistance to therapy, but the precise molecular mechanisms that regulate CAFs behavior are poorly understood. In this study, we utilized immortalized human pancreatic CAFs to investigate molecular pathways that control the matrix-remodeling and invasion-promoting activity of CAFs. We showed previously that palladin, an actin-associated protein, is expressed at high levels in CAFs of pancreatic tumors and other solid tumors, and also in an immortalized line of human CAFs. In this study, we found that short-term exposure of CAFs to phorbol esters reduced the number of stress fibers and triggered the appearance of individual invadopodia and invadopodial rosettes in CAFs. Molecular analysis of invadopodia revealed that their composition resembled that of similar structures (that is, invadopodia and podosomes) described in other cell types. Pharmacological inhibition and small interfering RNA knockdown experiments demonstrated that protein kinase C, the small GTPase Cdc42 and palladin were necessary for the efficient assembly of invadopodia by CAFs. In addition, GTPase activity assays showed that palladin contributes to the activation of Cdc42. In mouse xenograft experiments using a mixture of CAFs and tumor cells, palladin expression in CAFs promoted the rapid growth and metastasis of human pancreatic tumor cells. Overall, these results indicate that high levels of palladin expression in CAFs enhance their ability to remodel the extracellular matrix by regulating the activity of Cdc42, which in turn promotes the assembly of matrix-degrading invadopodia in CAFs and tumor cell invasion. Together, these results identify a novel molecular signaling pathway that may provide new molecular targets for the inhibition of pancreatic cancer metastasis.

Palladin contributes to invasive motility in human breast cancer cells.

Cancer metastasis involves multiple steps including detachment of the metastatic cells from neighboring cells, the acquisition of motility and invasion to other tissue. All of these steps require the reorganization of the actin cytoskeleton. In this study, we found that the protein palladin, a molecular scaffold with an important function in actin organization, is expressed at higher overall levels in tumors compared with benign breast tissue, and also expressed significantly higher in four invasive breast cancer cell lines when compared with four non-invasive cell lines. In addition, we found that palladin plays a key role in the formation of podosomes. Podosomes are actin-rich structures that function in adhesion and matrix degradation, and have been found in many invasive cell types. Our results show that phorbol ester treatment stimulated the formation of palladin-containing podosomes in invasive, but not in non-invasive cell lines. More importantly, palladin knockdown resulted in decreased podosome formation and a significant reduction in transwell migration and invasive motility. Palladin overexpression induced podosome formation in the non-invasive MCF7 cells, which are otherwise unable to form podosomes, suggesting that palladin plays a critical role in the assembly of podosomes. Overall, these results indicate that palladin overexpression contributes to the invasive behavior of metastatic cells.

The p115-interactive proteins GM130 and giantin participate in endoplasmic reticulum-Golgi traffic.

The transport factor p115 is essential for endoplasmic reticulum (ER) to Golgi traffic. P115 interacts with two Golgi proteins, GM130 and giantin, suggesting that they might also participate in ER-Golgi traffic. Here, we show that peptides containing the GM130 or the giantin p115 binding domain and anti-GM130 and anti-giantin antibodies inhibit transport of vesicular stomatitis virus (VSV)-G protein to a mannosidase II-containing Golgi compartment. To determine whether p115, GM130, and giantin act together or sequentially during transport, we compared kinetics of traffic inhibition. Anti-p115, anti-GM130, and anti-giantin antibodies inhibited transport at temporally distinct steps, with the p115-requiring step before the GM130-requiring stage, and both preceding the giantin-requiring stage. Examination of the distribution of the arrested VSV-G protein showed that anti-p115 antibodies inhibited transport at the level of vesicular-tubular clusters, whereas anti-GM130 and anti-giantin antibodies inhibited after the VSV-G protein moved to the Golgi complex. Our results provide the first evidence that GM130 and giantin are required for the delivery of a cargo protein to the mannosidase II-containing Golgi compartment. These data are most consistent with a model where transport from the ER to the cis/medial-Golgi compartments requires the action of p115, GM130, and giantin in a sequential rather than coordinate mechanism.

The membrane transport factor p115 recycles only between homologous compartments in intact heterokaryons.

Cytosolic proteins that participate in membrane traffic are assumed to be recruited from the cytosol onto specific membrane sites where they perform their function, and then released into cytosol before rebinding to catalyze another round of transport. To examine whether the ER to Golgi transport factor p115 recycles through release into a cytosolic pool, we formed heterokaryons between rat NRK and simian COS-7 cells and examined the dynamics of rat p115 transfer from the rat to the simian portion of the heterokaryon. The heterokaryons shared a common cytosolic pool, as shown by the efficient relocation of a cytosolic green fluorescent protein (GFP) from the COS-7 to the NRK part of the heterokaryon. Unexpectedly, even 24 h after cell fusion, rat p115 did not redistribute to the COS-7 part of the heterokaryon. This was not due to the inability of the rat p115 to associate with simian membranes since rat p115 expressed in COS-7 cells was efficiently targeted to and associated with simian Golgi complex. Furthermore, rat p115 associated with heterologous simian membranes after the NRK and COS-7 Golgi fused into a single chimeric structure. Our results indicate that p115 is not freely diffusible in intact cells and might remain tethered to membranes throughout its life cycle. These findings suggest that p115, and perhaps other cytosolic proteins involved in membrane traffic, recycle not by being released into cytosol, but in association with recycling membranes.

Binding relationships of membrane tethering components. The giantin N terminus and the GM130 N terminus compete for binding to the p115 C terminus.

By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathione S-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events.

Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera.

Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.

Contribution of breakdown in the regulation of mouse liver glutathione S-transferase subunits during protein depletion and re-feeding.

The in vivo radioactivity decay of glutathione S-transferase (GST) was observed in livers of normal-fed (N), protein-depleted (D), and re-fed mice (R), labelled with [35S]methionine. Half-lives in days at N, D and R, respectively, were: total GST, 1.7, 1.4, 4.3; Yb1-subunit, 2.0, 1.8, 3.3; Yc-subunit, 2.4, 1.0, 4.2; Yf-subunit, 1. 1, 2.1, 5.0. These findings, together with the fact that the proportions of GST-subunits depend on dietary protein, show that breakdown contributes differentially to control the content of each GST subunit. Data also indicate that GSTs are long-life proteins.

The membrane transport factor TAP/p115 cycles between the Golgi and earlier secretory compartments and contains distinct domains required for its localization and function.

The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al.; 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15 degreesC treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi. TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.

Protein depletion and refeeding change the proportion of mouse liver glutathione S-transferase subunits.

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.

[Active and inactive forms of cerebral cysticercosis. Study of 10 cases]. / Formas activas e inactivas de cisticercosis cerebral. Estudio de diez casos.

Ten cases of neuro-cysticercosis attended in our hospital over the last five years are presented. Six patients presented non-active parenchymatous calcifications of which four manifested epilepsy, one dementia caused by hydrocephalus and one other was asymptomatic. Two patients presented active intraparenchymatous cysts with a clinic of epilepsy. One patient presenting cisternal -ventricular-parenchymal involvement and another with ependymal -meningeal-parenchymal involvement, both suffered of psychiatric alterations. Ataxia and pyramidal deficits. The two patients with active parenchymal form were treated with Praziquantel obtaining a complete cyst remission (normal cranial Ot scan, in one case normal NMR) and neurological clinical symptoms. Two more patients treated with Praziquantel suffering ventricular and meningeal ependymal involvement presented little or no response to this treatment.