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.

To develop mechanically improved polylactide (PLA)-based sustainable polymers, a series of poly(lactide-bbutadiene) (PLA−PB) multiblock copolymers were synthesized in a two-step procedure: PLA−PB−PLA (LBL) triblock copolymers were prepared using ring-opening polymerization of D,L-lactide, followed by chain extension of LBL triblock polymers with toluene-2,4-diisocyanate (TDI) and terephthaloyl chloride (TCl). Molecular characterization revealed that the synthetic procedures yielded the desired triblock and multiblock copolymers with a composition range of 0.5 ≤ f PLA ≤ 0.9. Differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM) demonstrated nearly identical, well-ordered, morphologies in the homologous triblock and multiblock copolymer materials, in sharp contrast with the findings of a recent study involving poly(styrene-b-butadiene) (PS−PB) multiblock polymers. These results indicate a transition from classically ordered morphologies to a state of bicontinuous disorder for multiblocks containing ⟨n′⟩ ≥ 10, where ⟨n′⟩ is the average total number of blocks. Lamellae ( f PLA = 0.6) and cylinder ( f PLA = 0.7 and 0.8) forming PLA−PB multiblock copolymers exhibited dramatically enhanced mechanical properties compared to the corresponding LBL triblock copolymers. However, this toughening effect was not evident in samples containing a spherical morphology (f PLA = 0.9). These findings demonstrate a commercially viable approach to preparing sustainable plastics with competitive mechanical properties.

A common glycan alteration in transformed cells and human tumors is the highly elevated levels of N-linked ␤(1,6)glycans caused by increased transcription of Nacetylglucosaminyltransferase V (GnT-V). Here, we define the involvement of GnT-V in modulation of homotypic cell-cell adhesion in human fibrosarcoma HT1080 and mouse NIH3T3 cells. Increased GnT-V expression resulted in a significant decrease in the rates of calciumdependent cell-cell adhesion. Reduced cell-cell adhesion was blocked by function-blocking antibody against Ncadherin and abrogated by pre-treatment of cells with swainsonine, demonstrating the involvement of N-cadherin in the cell-cell adhesion and that changes in Nlinked ␤(1,6)glycan expression are responsible for the reduction in rates of adhesion, although this reduction could be mediated by the altered N-linked glycosylation of glycoproteins other than N-cadherin. Overexpression of GnT-V had no effect on the levels of cell surface expression of N-cadherin; however, it did cause a marked enhancement of both ␤(1,6) branching and poly-Nacetyllactosamine expression on N-cadherin. GnT-V overexpression resulted in decreased N-cadherin clustering on the cell surface induced by anti-N-cadherin antibody and affected the outside-in signal transduction pathway of ERK mediated by N-cadherin. Overexpression of GnT-V sensitized stimulation of tyrosine phosphorylation of catenins by growth factors and expression of v-src, which is consistent with its reduction of cell-cell adhesion. In vitro, GnT-V-overexpressing cells showed increased motility concomitant with increased phosphorylation of catenins. Moreover, GnT-V-deficient embryo fibroblasts from GnT-V homozygous null mice (GnT-V ؊/؊ ) express N-cadherin and showed significantly increased levels of N-cadherin-based cell-cell adhesion compared with those from GnT-V ؉/؊ mice. These levels of adhesion were inhibited significantly by transient expression of GnT-V, confirming the hypothesis that levels of GnT-V can regulate cadherin-associated homotypic cell-cell adhesion. Aberrant N-linked ␤(1,6) branching that occurs during oncogenesis can, therefore, lessen cell-cell adhesion, contributing to increased cellular motility and invasiveness.Changes in the expression of many cell surface adhesion receptors, including integrins, cadherins, CD44, and members of the immunoglobulin superfamily such as intercellular adhesion molecule, mediate cell-cell and cell-ECM 1 interactions that are clearly critical as cells undergo oncogenesis and show changes in motility and invasiveness (1-3). Recent studies demonstrate that aberrant glycosylation of several types of cell surface receptors results in dysfunctional intracellular signaling and altered cellular behavior. For example, mutations in an N-acetylglucosaminyltransferase, POMGnT-I, cause aberrant glycosylation of skeletal muscle dystrophin, resulting in dysfunctional neuromuscular junctions (4). Mutations in POMGnT-I have been shown to result in multiple phenotypes, some of which have been classified as "muscle-eye-b...

We report a synthetic method for the preparation of well-defined poly(styrene-b-butadiene) (PS-PB) n multiblock copolymers using a combination of living anionic polymerization and urethane based polycondensation along with an assessment of the associated microphase morphology and thermal and mechanical properties. The prepolymers, α,ω-dihydroxy polystyrene (HO-PS-OH) and α,ω-dihydroxy polybutadiene (HO-PB-OH), were prepared using a protected initiator, followed by deprotection and condensation with isophorone diisocyanate (IPDI) leading to the (PS-PB) n multiblock copolymers. Two types of block sequences were generated, one with random block sequences (PS-ran-PB) n and the second with alternating (PS-alt-PB) n block architectures. Size exclusion chromatography (SEC), 1 H nuclear magnetic resonance (NMR), and Fourier transform infrared spectrometry (FTIR) confirmed that the synthetic procedures yielded the desired products. Differential scanning calorimetry (DSC) revealed microphase separation and small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) demonstrated that the multiblock materials, containing between 69% and 85% by volume glassy PS, are characterized by a disordered bicontinuous-like morphology. Tensile tests demonstrated yielding followed by necking and an overall ductility that translates into much greater toughness than that typically found in glassy continuous SBS triblock copolymers.

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.tethers | GRASPs T he Golgi apparatus plays a central role in the processing, sorting, and secretion of various cargo molecules destined for various intracellular and extracellular destinations (1). In animal and plant cells, its unique structure of four to six stacked, roughly planar cisternae serves, among other things, as a platform to organize Golgi resident glycosyltransferases into distinct membrane-bound subcompartments (the cis-, medial-, and trans-Golgi cisternae) for proper and sequential posttranslational maturation of the transiting cargo proteins (2, 3).Although these characteristic features of Golgi morphology have drawn the attention of many researchers for many decades, the molecular mechanisms underlying them are still unclear (4). Pioneering functional reconstitution studies using a cell-free system in which Golgi stacks (but not ribbons) reassemble from mitotic extracts (5-8) yielded two classes of purified proteins, each clearly contributing to stacking: globular Golgi reassembly and stacking proteins (GRASPs; the homologous proteins GRASP65 and GRASP55) (7, 9) and the helical rod-like and partially homologous proteins Golgi matrix protein of 130 kDa (GM130) and Golgin of 45 kDa (Golgin45) (10, 11). One member of each family (GRASP65 and GM130) is located in the cis-most cisterna (7, 12), and another member of each family (GRASP5...