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Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that hosts complex signaling events and plays diverse roles in a variety of cellular processes. Secreted ER chaperones are capable of activating EGFR and its downstream transcription factor, STAT3. However, the molecular mechanism of these ER proteins triggering EGFR signaling is largely unknown. In this study, we identify KDEL receptor (KDELR) as a cell surface receptor for secreted ER chaperones and a co-receptor for EGFR. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and induces transcription of genes relevant to cell proliferation and migration. Taken together, our results suggest that secreted ER chaperones bind surface-expressed KDELR and transactivates EGFR-STAT3 signaling pathway.

KDEL receptor-1 maintains homeostasis in the early secretory pathway by capturing and retrieving ER-chaperones to the ER during heavy secretory activity. We have previously shown that a Golgi scaffolding protein (ACBD3) facilitates KDEL receptor localization at the Golgi via regulating cargo wave-induced cAMP/PKA-dependent signaling pathway. Unexpectedly, a fraction of the receptor is also known to reside in the plasma membrane as a stress response, although it is largely unknown exactly how KDEL receptor gets exported from the Golgi and travels to the PM. In this study, we sought to investigate the mechanism by which KDEL receptor gets exported from the Golgi en route to the PM and identified two crucial factors that greatly influence post-Golgi trafficking of KDEL receptor. We show here that ACBD3 depletion results in significantly increased trafficking of KDEL receptor to the PM via Rab4-positive tubular carriers emanating from the Golgi. Expression of constitutively activated Rab4 mutant (Q72L) increases surface expression of KDEL receptor up to 2~3-fold, whereas expression of GDP-locked Rab4 mutant (S27N) inhibits KDEL receptor localization to the PM. Importantly, KDELR trafficking from the Golgi to the PM is independent of PKA- and Src Kinase-mediated mechanism. Taken together, these results reveal that ACBD3 and Rab4-GTP are key players at the Golgi in regulating KDEL receptor trafficking to the cell surface.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

SUMOylation is a post-translational modification that attaches a ubiquitin-like modifier to target proteins and plays a number of roles throughout the cells, including transcriptional regulation and cellular stress response, etc. We have recently reported that a Golgi structural protein, Golgin45, is subjected to Tankyrase1-dependent poly (ADP-Ribosyl)ation, resulting in dynamic regulation of Golgin45 protein and membrane trafficking of glycosylating enzymes at the Golgi. However, exact mechanism that modulates Tankyrase activity on Golgin45 remained elusive. Here, we report that Golgin45 is a SUMOylated protein, and heat shock significantly increases Golgin45 SUMOylation, concomitantly leading to reduced PARylation. Importantly, while PML is required for Golgin45 SUMOylation by SUMO3, over-expression of a SUMOylation-deficient Golgin45 mutant inhibits the formation of PML-NBs under heat shock, revealing a role of SUMOylated Golgin45 in PML-NB formation in the nucleus. These results provide a new insight into stress-induced inter-organelle communications between the Golgi and the nucleus.

Retro-2 directly interacts with an ER exit site protein, Sec16A, inhibiting ER exit of a Golgi tSNARE, Syntaxin5, which results in rapid re-distribution of Syntaxin5 to the ER. Recently, it was shown that SARS-CoV-2 infection disrupts the Golgi apparatus within 6–12 hours, while its replication was effectively inhibited by Retro-2 in cultured human lung cells. Yet, exactly how Retro-2 may influence ultrastructure of the Golgi apparatus and its function have not been thoroughly investigated. In this study, we characterized the effect of Retro-2 treatment on Golgi function and its ultrastructure using electron microscopy and EM tomography. Our results indicate that Retro-2 treatment significantly alters protein glycosylation at the Golgi without affecting secretion of either small or large cargos. Ultra-structural study of the Golgi revealed rapid accumulation of COPI-like vesicular profiles in the perinuclear area and a partial disassembly of the Golgi stack under electron microscope within 3–5 hours, suggesting altered Golgi organization in these cells. Retro-2 treatment in cells depleted of GRASP65/55, the two well-known Golgi stacking proteins, induced complete and rapid disassembly of the Golgi into individual cisterna. Taken together, these results suggest that Retro-2 profoundly alters Golgi structure and function to a much greater extent than previously anticipated.

The unique stacked morphology of the Golgi apparatus had been a topic of intense investigation among the cell biologists over the years. We had previously shown that the two Golgin tethers (GM130 and Golgin45) could, to a large degree, functionally substitute for GRASP-type Golgi stacking proteins to sustain normal Golgi morphology and function in GRASP65/55-double depleted HeLa cells. However, compared to well-studied GM130, the exact role of Golgin45 in Golgi structure remains poorly understood. In this study, we aimed to further characterize the functional role of Golgin45 in Golgi structure and identified Golgin45 as a novel Syntaxin5-binding protein. Based primarily on a sequence homology between Golgin45 and GM130, we found that a leucine zipper-like motif in the central coiled-coil region of Golgin45 appears to serve as a Syntaxin5 binding domain. Mutagenesis study of this conserved domain in Golgin45 showed that a point mutation (D171A) can abrogate the interaction between Golgin45 and Syntaxin5 in pull-down assays using recombinant proteins, whereas this mutant Golgin45 binding to Rab2-GTP was unaffected in vitro. Strikingly, exogenous expression of this Syntaxin5 binding deficient mutant (D171A) of Golgin45 in HeLa cells resulted in frequent intercisternal fusion among neighboring Golgi cisterna, as readily observed by EM and EM tomography. Further, double depletion of the two Syntaxin5-binding Golgin tethers also led to significant intercisternal fusion, while double depletion of GRASP65/55 didn’t lead to this phenotype. These results suggest that certain tether-SNARE interaction within Golgi stack may play a role in inhibiting intercisternal fusion among neighboring cisternae, thereby contributing to structural integrity of the Golgi stack.

Ebola virus is a member of Filoviridae family of viruses that causes fetal hemorrhagic fever in human. Matrix protein VP40 of the Ebola virus is involved in multiple stages of viral maturation processes. In order to fully understand the interacting partners of VP40 in host cells, we applied proximity-dependent biotin-identification (BioID) approach to systematically screen for potential proteins at different time points of VP40 expression. By immunoprecipitation and subsequent proteomics analysis, we found over 100 candidate proteins with various cellular components and molecular functions. Among them, we identified Rab14 GTPase that appears to function at the late stage of VP40 expression. Imaging studies demonstrated that VP40 and Rab14 have substantial colocalization when expressed in HeLa cells. Overexpression of the dominant-negative Rab14(S25N) diminished the plasma membrane (PM) localization of VP40. In addition, we found that secreted VP40 protein can be endocytosed into Rab14 positive compartments. In summary, our study provides evidence that Rab14 is a novel regulator of the intracellular trafficking of Ebola virus matrix protein VP40 in HeLa cells.

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

Acyl-CoA-binding domain-containing 3 (ACBD3) is a multi-functional scaffolding protein, which has been associated with a diverse array of cellular functions, including steroidogenesis, embryogenesis, neurogenesis, Huntington’s disease (HD), membrane trafficking, and viral/bacterial proliferation in infected host cells. In this review, we aim to give a timely overview of recent findings on this protein, including its emerging role in membrane domain organization at the Golgi and the mitochondria. We hope that this review provides readers with useful insights on how ACBD3 may contribute to membrane domain organization along the secretory pathway and on the cytoplasmic surface of intracellular organelles, which influence many important physiological and pathophysiological processes in mammalian cells.

We analyzed the destruction patterns of a turbo-molecular pump (TMP) resulting from its sudden exposure of a foreline to the atmospheric pressure due to a destruction of the foreline connecting clamp of an ICP dry etcher for 300 mm wafers during high-vacuum operation (5×10 ?6 Torr). Unlike in the case of view port’s breakage, the TMP’s rotor module was crashed inside the chamber. The primary damage resulted from the collision of the blades and stators, and the secondary damage resulted from the breaking of the rotor - driving shaft assembly. The fixing screws of the rotor and axial shaft were bent and broken when the TMP controller output the maximum current even after the crash event. Electrical power consumption analysis of the TMP power controller confirmed it. The stress distributions were analyzed by a finite element method using CFD-ACE+ multi physics software. Rotating inertia of each parts and kinetic energies were calculated as well. 68% of the rotational kinetic energy is deposited by the rotor - shaft module.

Acyl-CoA-binding domain-containing 3 (ACBD3) is a multi-functional scaffolding protein, which has been associated with a diverse array of cellular functions, including steroidogenesis, embryogenesis, neurogenesis, Huntington’s disease (HD), membrane trafficking, and viral/bacterial proliferation in infected host cells. In this review, we aim to give a timely overview of recent findings on this protein, including its emerging role in membrane domain organization at the Golgi and the mitochondria. We hope that this review provides readers with useful insights on how ACBD3 may contribute to membrane domain organization along the secretory pathway and on the cytoplasmic surface of intracellular organelles, which influence many important physiological and pathophysiological processes in mammalian cells.

Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.

Adaptor protein-1 (AP-1) is recruited onto the trans-Golgi network via binding to Arf-1·GTP, cargo-sorting signals and phosphoinositides, where it orchestrates the assembly of clathrin-coated vesicular carriers that transport cargo molecules to endosomes. Here we show that cytosolic AP-1 polymerizes when recruited onto enriched Golgi membranes and liposomes containing covalently attached cargo-sorting signal peptides. Incubation of cytosolic or purified AP-1 with soluble sorting signal peptides also resulted in AP-1 polymerization, showing that Arf-1·GTP and membranes are not required for this process. We propose that cargo-induced polymerization of AP-1 contributes to stabilization of the coat complex in the formation of clathrin-coated buds.

Understanding of electrophoretic mobility and zeta potential is an important issue in charge characteristics of proteins for proteomics research. By exploring the electrostatic and electrokinetic principles, we presented a framework that would allow rigorous interpretations of surface zeta potential and effective charge for highly charged conditions holding the full range of buffer pH. Together with an analytic expression by considering the Henry's formula with Debye–Huckel ansatz, the zeta potential at the protein surface was evaluated from an integral expression for the electrophoretic mobility of a spherical particle on the basis of nonlinear Poisson–Boltzmann equation. Subsequently, the effective protein charge was evaluated from the corresponding relationships between potential distribution and surface charge, with accounting for the presence of an electric double layer. Applying the microchip electrophoresis, the experimental results of mobility data were obtained for model proteins with bovine serum albumin and ovalbumin in different pH of buffer solution. To illustrate the usefulness of our considerations, experimental data of electrophoretic mobility available in the literature are also included. It is evident that the results by linear correlations are identified to overestimate the zeta potential, while they underestimate the effective charge. The discrepancy between linear and nonlinear correlations is trivial for the zeta potential less than 25.69 mV, however, it fairly increases with increasing the absolute value of zeta potential.

The adaptor protein AP-1 is the major coat protein involved in the formation of clathrin-coated vesicles at the trans-Golgi network. The prevailing view is that AP-1 recruitment involves coincident binding to multiple low-affinity sites comprising adenosine diphosphate ribosylation factor 1 (Arf-1)–guanosine triphosphate (GTP), cargo sorting signals, and phosphoinositides. We now show that binding of cargo signal peptides to AP-1 induces a conformational change in its core domain that greatly enhances its interaction with Arf-1–GTP. In addition, we provide evidence for cross talk between the dileucine and tyrosine binding sites within the AP-1 core domain such that binding of a cargo signal to one site facilitates binding to the other site. The stable association of AP-1 with Arf-1–GTP, which is induced by cargo signals, would serve to provide sufficient time for adaptor polymerization and clathrin recruitment while ensuring the packaging of cargo molecules into the forming transport vesicles.

O-mannosyl-linked glycans constitute a third of all brain O-linked glycoproteins, and yet very little is understood about their functions. Several congenital muscular dystrophies with central nervous system defects are caused by genetic disruptions in glycosyltransferases responsible for the synthesis of O-mannosyl glycans. The glycosyltransferase GnT-Vb, also known as GnT-IX, is expressed abundantly in the brain and testis and is proposed to be the enzyme that branches O-mannosyl-linked glycans. In this study, we show in a human neuronal model that GnT-Vb expression enhances neurite outgrowth on laminin. GnT-Vb has been shown to perform both N-linked and O-mannosyl-linked glycosylation. To determine if the effect on neurite outgrowth was due to N-linked or O-mannosyl-linked glycosylation by GnT-Vb we suppressed the expression of glycosyltransferases important for the elongation of both N-linked and O-mannosyl-linked glycans using RNA interference. Our results suggest that GnT-Vb and PomGnT1, enzymes involved in the O-mannosyl glycosylation pathway, play an active role in modulating integrin and laminin-dependent adhesion and migration of human neuronal cells.

N-acetylglucosaminyltransferase VB (GnT-VB, -IX) is a newly discovered glycosyltransferase expressed exclusively in high levels in neuronal tissue during early development. Its homolog, GnT-V, is expressed in many tissues and modulates cell-cell and cell-matrix adhesion. The ability of GnT-VB to regulate cell-matrix interactions was initially investigated using the rat pheochromocytoma PC12 neurite outgrowth model. PC12 cells stably transfected with GnT-VB consistently showed an enhanced rate of nerve growth factor (NGF)-induced neurite outgrowth on collagen and laminin substrates. Levels of TrkA receptor phosphorylation and downstream ERK activation induced by NGF were not influenced by GnT-VB expression. No significant difference was observed in the rate of neurite outgrowth when cells were cultured on non-coated culture dishes, indicating that integrin-ECM interaction is required for the stimulatory effects. Neurite outgrowth induced by manganese-dependent activation of beta1 integrin on collagen and laminin substrates, however, showed a significant increase in neurite length for the PC12/GnT-VB cells, compared with control cells, suggesting that the enhancement is most likely mediated by alteration of beta1 integrin-ECM interaction by GnT-VB. These results demonstrate that GnT-VB expression can modulate the rate of neurite outgrowth by affecting beta1 integrin-ECM interaction.

An N-linked glycan often increased during oncogenic transformation contains beta(1,6)-linked GlcNAc, synthesized by the N-acetylglucosaminyltransferase V (GnT-V). The progression of polyoma middle T-antigen oncoprotein-induced mammary carcinomas in GnT-V null mice was significantly retarded compared with that observed in wild-type mice. The matrix adhesion of mouse embryonic fibroblasts (MEF) from GnT-V null and wild-type mice was investigated to understand the mechanism by which deletion of GnT-V could retard tumor progression. GnT-V null MEF displayed enhanced adhesion to and spreading on fibronectin-coated plates with concomitant inhibition of cell migration. GnT-V null MEF also showed increased focal adhesion kinase tyrosine phosphorylation, consistent with decreased cell motility on fibronectin-coated plates. Expression of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes. The alpha5beta1 fibronectin receptors exhibited increased clustering on the null MEF cell surfaces, consistent with previous studies that observed less integrin clustering in cells overexpressing GnT-V. Most surprisingly, GnT-V null MEF displayed increased expression levels of both alpha5 and beta1 subunits in lysates and on the cell surface. Increased alpha5beta1 expression in the null MEF was because of increased alpha5beta1 transcript levels that declined after re-expression of GnT-V cDNA, confirming that increased alpha5beta1 expression in null MEF was because of changes in GnT-V expression. The increased null MEF transcripts were shown to be caused at least in part by increased integrin promoter activity. Moreover, increased alpha5beta1 integrin transcripts in GnT-V null MEF were not due to a differential response to fibronectin; rather, they appeared to be mediated by activation of a protein kinase C signaling pathway. These results demonstrate that deletion of MEF GnT-V resulted in enhanced integrin clustering and activation of alpha5beta1 transcription by protein kinase C signaling, which in turn up-regulated levels of cell surface alpha5beta1 fibronectin receptors that resulted in increased matrix adhesion and inhibition of migration.

Vacuolar H(+)-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H(+)-ATPase via one of its transmembrane domains, TMD4, strongly associates with beta(1) integrin, affecting beta(1) integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of beta(1) integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose alpha(5)beta(1) integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of beta(1) integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of beta(1) integrin (premature form) and, consequently, the level of the 130-kDa form of beta(1) integrin (mature form). Further experiments showed that the normal levels of association between the premature beta(1) integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of beta(1) integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits beta(1) integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional beta(1) integrin.

UDP-N-acetylglucosamine:alpha(1,6)-D-mannoside beta(1,6)-N-acetylglucosaminyltransferase (GnT-V, Mgat5) functions in the biosynthesis of N-linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct enzyme with related substrate specificity, termed GnT-VB, which is predicted to have 53% similarity to the original amino acid sequence of GnT-V(A). Transient expression of GnT-VB cDNA in COS7 cells yielded significant increases of activity toward GnT-VA acceptors, including synthetic saccharides and N-linked glycopeptides, with some differences in specificity. Unlike GnT-VA, GnT-VB required divalent cation for full activity. EST databases showed expression of a 6 bp (+) splice isoform of GnT-VB; when expressed, this enzyme showed significantly reduced activity. CHO Lec4 cells, which do not express GnT-VA or B activity, lack synthesis of the N-linked beta(1,6) branch, and do not bind L-phytohemagglutinin (L-PHA), were transfected with GnT-VB or GnT-VA; both then bound significant amounts of L-PHA, demonstrating that both enzymes synthesized N-linked beta(1,6) branched glycans in vivo. Real-time polymerase chain reaction results showed that GnT-VB mRNA was highly expressed in brain and testis, with lesser levels in other tissues, while human GnT-VA showed a more general expression, but with low levels in brain and no expression in skeletal muscle.

For the efficient virtual try-out loop, geometric change and bending angles have been compensated during last 20 years. This approach was based on some restrictions like pure bending, plane strain state and isotropic behavior. For more complex forming processes, this has been applied without reviewing this limitation. Analytical force consideration to reduce the amount of springback is also another idea to compensate geometrical displacement adjustment efficiently. In other view, the springback prediction accuracy is also one major topic with various material model developments. All these topics are absolutely of high importance in order to increase springback accuracy and effective compensation to help reduce the trials efforts. But the focus should not only be on these advances issues since the basics must be right as well. Based on our long term experiences in simulation and several project outcomes covered 20 different parts during last 2 years, we will present our experience and investigations in order to geometrically compensate forming process. It is emphasized that we did not just simulate the springback but the compensated surfaces where brought into the real tools. So, the experiences are not only based on numerical analysis but of all this part a physical tryout has been performed as well. The experiences are consolidated in a set of principles of robust springback compensation. These principles will be illustrated and explained at an example part, a B-pillar upper reinforcement. Indeed, it is can be seen that that compensation is a straight forward activity. However, our experiences have shown that it is only a straight forward activity in case certain ‘boundary conditions are fulfilled. We will discuss some of this boundary condition using the B-pillar upper example. Through this study, basic requirements for successful springabck compensation, the full scope of simulation range, set-up the nominal springback simulation and robustness of forming process will be clearly described to help understanding practical meanings of springback accuracy and strategic compensation.

Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAcbeta(1,6)Man branching, we utilized specific siRNA to selectively knockdown GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in the attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced downregulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor downregulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause the inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced downregulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by the expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by the treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes.

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma1 or alpha/sigma2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta1/mu1 and beta2/mu2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma1 and alpha/sigma2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma1 hemicomplex, whereas minimally affecting binding to alpha/sigma2. There was an excellent correlation between binding to the alpha/sigma2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.

Altered expression of cell surface N-linked oligosaccharides is associatedwith the oncogenic transformation of many types of animal cells. One of the most common forms of glycosylation in transformed cells and human tumors is the highly elevated beta1,6 branching of N-linked oligosaccharides caused by increased transcription of N-acetylglucosaminyltransferase V (GnT-V). To characterize the effects of increased beta1,6 branching on cell-matrix adhesion-mediated phenotypes, human fibrosarcoma HT1080 cells were transfected with retroviral systems encoding GnT-V that used both noninducible and tetracycline-inducible promoters. Increased GnT-V expression resulted in a25% inhibition of cell attachment to and a50% inhibition of cell spreading on fibronectin. Both cell adhesion and spreading were suppressed by function-blocking antibodies specific for the alpha(5) and beta(1) integrin subunits of the fibronectin receptor. Cell migration toward fibronectin and invasion through Matrigel were both substantially stimulated in cells with induced expression of GnT-V. Induction of GnT-V had no effect on the level of cell surface expression of alpha(5) and beta(1) integrin subunits but did result in a more diffuse staining of the alpha(5) and beta(1) integrin subunits on the cell surface, suggesting that inhibition of integrin clustering may be causing these cells to be less adhesive and more motile. Surprisingly, there was no detectable expression of N-linked beta1,6 branching on the alpha(5) subunit purified from HT1080 cells before and after induction of GnT-V; by contrast, however, the beta(1) subunit showed a basal level of beta1,6 branching that was greatly increased after induction of GnT-V. These results suggest that changes in N-linked beta1,6 branching that occur during oncogenesis alter cell-matrix adhesion and migration by modulating integrin clustering and subsequent signal transduction pathways. These effects most likely result from altered N-linked carbohydrate expression on the beta(1) integrin subunit.

This study is focused on the practical application process for the product design topredict the serviceability, reliability, and manufacturability of structures and components.In order to prevent the failure, assessment program must be developed efficiently toaccount for the reason of failure. As for the failure mechanism, many experimentalmethods have been developed and standardized, and the computational finite elementapproaches have introduced for prediction of fracture. These computational finite elementapproaches always need implementing the validation process with corresponding testprocedures. By the virtue of high performance computing technology and advancedsoftware development, complicate structural geometry subjected to complicate loadingconditions can be simulated and analyzed with a good accuracy. But finite elementapproaches have been challengingly used in failure prediction on the dynamic and fatigueloading, and it shows less 50% accuracy in most cases. The high frequency vibration ornumerical noise is one of concerns during the explicit dynamic simulations for accuracy.2The misinterpretation with experimental results is also one of concerns and makes usgone in to a precedent of invisible error in the complicated dynamic loadings. It is saidthat the numerical simulation modeling is strongly associated with experimental methodand the new analysis process of dynamic failure must be developed by through thesimultaneous and progressive steps.In this study, therefore, the aim is to increase the accuracy on prediction of dynamic andfatigue failure with respect to practical structure and components. The full analysisprocesses comprised calibration, validation and prediction phases will be presented in thisstudy, and associated experimental approaches are also presented. Important aspect ofthis failure assessment process is newly to introduce the latest continuum damagemechanics model coupled with conventional finite element method. Predicting theimplications of structural deterioration for mechanical integrity is the goal of continuumdamage mechanics and it is purposed to increase the accuracy of failure prediction.Continuum damage mechanics framework has advances to the engineering point ofview how they use it simply like a tool with wide application in industry.Since failure is a complex nonlinear process dealing the creation and growth of microvoidsor micro-cracks, the predicted material behavior can be sensitive to parameters.Their appropriate identification is a key for accuracy of prediction and completedevelopment of analysis process.After exhibition of conventional full analysis process for dynamic failure of aluminumcasting alloy in the second chapter, introductory conclusion focus on how the continuumdamage mechanics concept is inducted into the dynamic fracture to be consideredmanufacturing process effects such as solidification time. The remaining chapters present3details of analysis process focusing dynamic fracture toughness KId of aluminum matrixcomposites on specimen level, fracturing of semi assembly leveled structure withaluminum honeycomb sandwich panel and fatigue failure with steel alloy. Through thisseveral chapters, the complete analysis process using one integrated material model basedon continuum damage mechanics progress in how to simulate the failure of real structuresubjected to different loadings such as dynamic and fatigue loading.This study is originally proposed to engineering of failure analysis processes throughcalibration, validation and prediction process and will be one progress on the accuracy offailure prediction.It should appeal to wide range of engineering concerned with lifetime prediction andwith the dynamic failure resistant design of structure, components and process ofmanufacturing. But this study limits on the isotropic materials and simple mode I crackpropagation without bifurcation of crack and can not handle the discrete crack initiationand propagation, because this study based on the continuum mechanics and isotropicconstitutive law. The extended finite element method (X-FEM) of the modeling of crackdiscontinuities encourages to be considered as new methodology of the warrantedaccuracy with a special discontinuous functions and asymptotic crack-tip displacementfields for future study., 2009