bluuebunny/crossref_metadata_2025_split huggingface dataset:
Yeast ER-Golgi v-SNAREs Bos1p and Bet1p differ in steady-state localization and targeting
Authors: Ossipov Dmitri , Schröder-Köhne Stephan , Schmitt Hans Dieter 
Published: April 2021

ABSTRACT
Vesicle specific SNAP receptors (v-SNAREs) Bos1p and Bet1p are involved in targeting of anterograde vesicles between the endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors that influence the targeting of these proteins, α-factor tagged versions of Bos1p and Bet1p were employed. The α-factor can be cleaved off by the Kex2p protease as soon as the hybrid proteins reach the late Golgi compartment. The data obtained by monitoring of Kex2p cleavage, by immunofluorescence microscopy and cell fractionation showed that Bos1-α and Bet1-α have different cellular localization and dynamics. Bos1-α is an ER protein, which recycles between the Golgi and the ER in COPI-dependent manner. Bet1-α is an early Golgi protein and it does not change its localization under conditions when other recycling Golgi proteins can be trapped in the ER.
Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane
Authors: Romisch Karin 
Published: April 2021

ABSTRACT
Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.
Essential role of biliary glycoprotein (CD66a) in morphogenesis of the human mammary epithelial cell line MCF10F
Authors: Huang Jie , Hardy John D. , and Yanyu Sun , Shively John E. 
Published: April 2021

ABSTRACT
Normal mammary epithelial cells express the cell surface protein biliary glycoprotein (BGP or CD66a) in a polarized manner, suggesting that this protein may play a role in the formation of mammary acini. In order to test this hypothesis, we interrupted the expression of BGP in the mammary epithelial line MCF10F when cultured in or on Matrigel, a source of extracellular matrix (ECM). When analyzed by immunofluorescence confocal microscopy, the BGP staining is confined to the lumenal surface and colocalizes with actin.
Sequential scanning electron microscopy demonstrates that the MCF10F cells migrate to form clusters, followed by apoptotic cell death within the center, resulting in lumen formation. Transmission electron micrographs reveal the presence of tight junctions and desmosomes between the cells, microvilli along the lumenal surface, and typical apoptotic bodies within the lumen. When the MCF10F cells are transfected with the BGP antisense gene and grown in Matrigel, they exhibit reduced acini formation (12% and 20%) compared to untransfected cells (52%) or to cells transfected with vector only (62%). Acini formation is also significantly reduced when MCF10F cells grown in Matrigel are treated with anti-BGP antibody (18% at 100 pg/ml), or recombinant soluble BGP (18% at 0.4 pM). In contrast, the BGP-negative MCF7 breast tumor cell line, which does not form acini when grown in matrigel, exhibits &gt;60% cell death with the occasional formation of acini, when transfected with the BGP sense gene and grown in Matrigel. These results support the hypothesis that BGP plays a role in the normal differentiation program of mammary epithelial cells, indicating that its expression is essential to the formation of the lumen. Furthermore, and as shown by others, the differentiation program depends on the presence of ECM. The lack of expression of BGP in the MCF7 breast cancer cell line suggests that the downregulation of BGP expression confers a growth advantage to these cells in ECM. In addition, we found that the MCF10F cells could be separated into a BGP-positive epithelial fraction (MCF10F-e), and a BGP-negative myoepithelial fraction (MCF10F-m). When the myoepithelial cell-enriched fraction is grown on Matrigel, web-like structures are formed. These cells have a typical spindle shape cell morphology and express keratin, oc-smooth muscle actin and vimentin, markers of the myoepithelial cell phenotype. When MCF10F-m cells are treated with IFNy, they express CEA (carcinoembryonic antigen) but not BGP. Since breast carcinomas, especially in situ carcinomas, express CEA, this finding may suggest a heretofore unappreciated relationship between myoepithelial cells and breast cancer.
In vivo angiogenic activity of urokinase: role of endogenous fibroblast growth factor-2
Authors: Ribatti Domenico , Leali Daria , Vacca Angelo , Giuliani Roberta , Gualandris Anna , Roncali Luisa , Nolli Maria Luisa , Presta Marco 
Published: April 2021

ABSTRACT
In vitro experimental evidences suggest that the proteolytic degradation of the extracellular matrix (ECM) by activation of the urokinase-type plasminogen activator (uPA)/plasmin system may affect growth factor activity and bioavailability. However, no direct in vivo observations were available to support this hypothesis. Here we demonstrate that endothelial GM 7373 cells overexpressing human uPA (uPA-R5 cells) cause the release of 125I-labeled fibroblast growth factor-2 (FGF2) from endothelial ECM in a plasmin-dependent manner. Accordingly, uPA-R5 cells are angiogenic in vivo when applied on the top of the chorioallantoic membrane (CAM) of the chick embryo. In contrast, mock-transfected Neo2 cells are unable to release ECM-bound 125I-FGF2 and are poorly angiogenic. Neovascularization elicited by uPA-R5 cells is significantly reduced by neutralizing anti-FGF2 antibodies to values similar to those observed in Neo2 cell-treated CAMs. Accordingly, purified human uPA stimulates neovascularization of the CAM in the absence of an inflammatory response. The angiogenic activity of uPA is significantly inhibited by neutralizing anti-FGF2 antibodies or by pretreatment with phenylmethylsulfonyl fluoride. The non-catalytic, receptor-binding aminoterminal fragment of uPA is instead non angiogenic. Taken together, the data indicate that uPA is able to induce angiogenesis in vivo via a plasmin-dependent degradation of ECM that causes the mobilization of stored endogenous FGF2.
E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics
Authors: Faire Kathleen , Waterman-Storer Clare M. , Gruber Dorota , Masson Danièle , Salmon E. D. , Chloë Bulinski J. 
Published: April 2021

ABSTRACT
Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2×, 3×, or 4×GFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2×GFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2×GFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.
Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXФ targeting signals
Authors: Gough Nancy R. , Zweifel Mark E. , Martinez-Augustin Olga , Aguilar Ruben C. , Bonifacino Juan S. , Fambrough Douglas M. 
Published: April 2021

ABSTRACT
A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the m2 and m3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.
Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations
Authors: Quiñones Beatriz , Riento Kirsi , Olkkonen Vesa M. , Hardy Stephen , Bennett Mark K. 
Published: April 2021

ABSTRACT
The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2’), 2C (2’’) and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.
Molecular map of the desmosomal plaque
Authors: North Alison J. , Bardsley William G. , Hyam Janine , Bornslaeger Elayne A. , Cordingley Hayley C. , Trinnaman Brian , Hatzfeld Mechthild , Green Kathleen J. , Magee Anthony I. , Garrod David R. 
Published: April 2021

ABSTRACT
Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the ‘a’ form of
desmocollin overlaps with plakoglobin and desmoplakin, the shorter ‘b’ form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled.
Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.
XMAP230 is required for normal spindle assembly in vivo and in vitro
Authors: Cha Byeong-Jik , Cassimeris Lynne , Gard David L. 
Published: April 2021

ABSTRACT
XMAP230 is a high molecular mass microtubule-associated protein isolated from Xenopus oocytes and eggs, and has been recently shown to be a homolog of mammalian MAP4. Confocal immunofluorescence microscopy revealed that XMAP230 is associated with microtubules throughout the cell cycle of early Xenopus embryos. During interphase XMAP230 is associated with the radial arrays of microtubules and midbodies remaining from the previous division. During mitosis, XMAP230 is associated with both astral microtubules and microtubules of the central spindle. Microinjection of affinity-purified anti-XMAP230 antibody into blastomeres severely disrupted the assembly of mitotic spindles during the rapid cleavage cycles of early development. Both monopolar half spindles and bipolar spindles were assembled from XMAP230-depleted extracts in vitro. However, spindles assembled in XMAP230-depleted extracts exhibited a reduction in spindle width, reduced microtubule density, chromosome loss, and reduced acetylation of spindle MTs. Similar defects were observed in the spindles assembled in XMAP230-depleted extracts that had been cycled through interphase. Depletion of XMAP230 had no effect on the pole-to-pole length of spindles, and depletion of XMAP230 from both interphase and M-phase extracts had no effect on the rate of microtubule elongation. From these results, we conclude that XMAP230 plays an important role in normal spindle assembly, primarily by acting to stabilize spindle microtubules, and that the observed defects in spindle assembly may result from enhanced microtubule dynamics in XMAP230-depleted extracts.
A linking function for the cellulose-binding protein SP85 in the spore coat of Dictyostelium discoideum
Authors: Zhang Yunyan , Zhang Ping , West Christopher M. 
Published: April 2021

ABSTRACT
SP85 is a multidomain protein of the Dictyostelium spore coat whose C-terminal region binds cellulose in vitro. To map domains critical for localizing SP85 and for binding to other proteins in vivo, its N- and C-terminal regions, and a hybrid fusion of the N- and C-regions, were expressed in prespore cells. Immunofluorescence showed that only the N-terminal region and the N/C-hybrid accumulated in prespore vesicles, where coat proteins are normally stored prior to secretion. In contrast, only the C-terminal region and N/C-hybrid were incorporated into the coat after secretion. To determine if SP85 is important for the incorporation of other coat proteins, an SP85-null strain was created and found to mislocalize the coat protein SP65 to the interspore matrix. In vitro binding studies demonstrated that the SP85 C-terminal region bound SP65 and cellulose simultaneously, and SP65 incorporation was rescued in vivo by the C-terminal region. SP85-null spores showed increased latent permeability to a fluorescent lectin probe and accelerated germination times, and decreased buoyant density of their coats, suggesting that coat barrier functions were compromised. Dominant negative reductions in barrier functions also resulted from expression of the SP85 terminal regions, suggesting that a linking activity was important for SP85’s function. Thus, separate domains of SP85 specify prespore vesicle compartmentalization and coat incorporation, and additional domains link SP65 to the coat and simultaneously interact with other binding partners which contribute to coat barrier functions.
Characterization of the human Suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli
Authors: Stone Donna M. , Murone Maximilien , Luoh Shiuh-Ming , Ye Weilan , Armanini Mark P. , Gurney Austin , Phillips Heidi , Brush Jennifer , Goddard Audrey , Sauvage Frederic J. de , Rosenthal Arnon 
Published: April 2021

ABSTRACT
Drosophila Suppressor of fused (Su(fu)) encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Here we describe the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) (hSu(fu)). Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms were expressed in multiple embryonic and adult tissues, and exhibited a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus mapped to chromosome 10q24-q25, a region which is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) was found to repress activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homologue. Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway.
Although calnexin is essential in S. pombe, its highly conserved central domain is dispensable for viability
Authors: Elagöz Aram , Callejo Mario , Armstrong John , Rokeach Luis A. 
Published: April 2021

ABSTRACT
In mammalian cells, the calnexin/calreticulin chaperones play a key role in glycoprotein folding and its control within the endoplasmic reticulum (ER), by interacting with folding intermediates via their monoglucosylated glycans. This lectin activity has been mapped in mammalian calnexin/calreticulin chaperones to the central region, which is a highly conserved feature of calnexin/calreticulin molecules across species. The central domain has also been implicated in Ca2+ binding, and it has been proposed to be involved in the regulation of calcium homeostasis in the ER. Herein, we show that although the Schizosaccharomyces pombe calnexin is essential for viability, cells lacking its 317-amino-acid highly conserved central region are viable under normal growth conditions. However, the central region appears to be necessary for optimal growth under high ER-stress, suggesting that this region is important under extreme folding situations (such as DTT and temperature). The minimal length of calnexin required for viability spans the C-terminal 123 residues. Furthermore, cells with the central domain of the protein deleted were affected in their morphology at 37°C, probably due to a defect in cell wall synthesis, although these mutant cells exhibited the same calcium tolerance as wild-type cells at 30°C.
Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications for microfilament reorganization during apoptosis
Authors: Sgorbissa Andrea , Benetti Roberta , Marzinotto Stefania , Schneider Claudio , Brancolini Claudio 
Published: April 2021

ABSTRACT
Apoptosis is characterized by proteolysis of specific cellular proteins by a family of cystein proteases known as caspases. Gas2, a component of the microfilament system, is cleaved during apoptosis and the cleaved form specifically regulates microfilaments and cell shape changes. We now demonstrate that Gas2 is a substrate of caspase-3 but not of caspase-6. Proteolytic processing both in vitro and in vivo is dependent on aspartic residue 279. Gas2 cleavage was only partially impaired in apoptotic MCF-7 cells which lack caspase-3, thus indicating that different caspases can process Gas2 in vivo. In vitro Gas2 was processed, albeit with low affinity, by caspase-7 thus suggesting that this caspase could be responsible for the incomplete Gas2 processing observed in UV treated MCF-7 cells. In vivo proteolysis of Gas2 was detected at an early stage of the apoptotic process when the cells are still adherent on the substrate and it was coupled to the specific rearrangement of the microfilament characterizing cell death. Finally we also demonstrated that Gas2 in vitro binds to F-actin, but this interaction was unaffected by the caspase-3 dependent proteolytic processing.