bluuebunny/crossref_metadata_2025 huggingface dataset:
VE-cadherin and desmoplakin are assembled into dermal microvascular endothelial intercellular junctions: A pivotal role for plakoglobin in the recruitment of desmoplakin to intercellular junctions
Authors: Kowalczyk Andrew P. , Navarro Pilar , Dejana Elisabetta , Bornslaeger Elayne A. , Green Kathleen J. , Kopp Daniel S. , Borgwardt Jeffrey E.
Published: April 2021

Vascular endothelial cells assemble adhesive intercellular junctions comprising a unique cadherin, VE-cadherin, which is coupled to the actin cytoskeleton through cytoplasmic interactions with plakoglobin, β-catenin and α-catenin. However, the potential linkage between VE-cadherin and the vimentin intermediate filament cytoskeleton is not well characterized. Recent evidence indicates that lymphatic and vascular endothelial cells express desmoplakin, a cytoplasmic desmosomal protein that attaches intermediate filaments to the plasma membrane in epithelial cells. In the present study, desmoplakin was localized to intercellular junctions in human dermal microvascular endothelial cells. To determine if VE-cadherin could associate with desmoplakin, VE-cadherin, plakoglobin, and a desmoplakin amino-terminal polypeptide (DP-NTP) were co-expressed in L-cell fibroblasts. In the presence of VE-cadherin, both plakoglobin and DP-NTP were recruited to cell-cell borders. Interestingly, β-catenin could not substitute for plakoglobin in the recruitment of DP-NTP to cell borders, and DP-NTP bound to plakoglobin but not β-catenin in the yeast two-hybrid system. In addition, DP-NTP colocalized at cell-cell borders with α-catenin in the L-cell lines, and endogenous desmoplakin and α-catenin colocalized in cultured dermal microvascular endothelial cells. This is in striking contrast to epithelial cells, where desmoplakin and α-catenin are restricted to desmosomes and adherens junctions, respectively. These results suggest that endothelial cells assemble unique junctional complexes that couple VE-cadherin to both the actin and intermediate filament cytoskeleton.
Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPγS in vitro
Authors: Nickel Walter , Malsam Jörg , Gorgas Karin , Ravazzola Mariella , Jenne Nicole , Helms J. Bernd , Wieland Felix T.
Published: April 2021

On the basis of the cell surface protein CD8 we have constructed reporter molecules for both anterograde and retrograde transport from the Golgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of the viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CD8-LT). Due to this masking of the KKXX retrieval signal CD8-LT is transported to the cell surface. Since the KKXX motif is joined to the myc epitope via a thrombin cleavage site, CD8-LT in isolated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro. A CHO cell line stably expressing CD8-LT was generated and used for the isolation of Golgi membranes. These membranes were shown to contain CD8-LT en route to the cell surface. By addition of thrombin, CD8-LT could be efficiently converted into CD8-ST, and this allows us to study the sorting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated in vitro from Golgi membranes containing either CD8-LT or CD8-ST. When the incubation was performed in the presence of GTP, both CD8-LT and CD8-ST were packaged into COPI-coated vesicles. However, COPI-coated vesicles generated in the presence of the slowly hydrolyzable analogue of GTP, GTPγS contained strikingly lower amounts of CD8-LT and CD8-ST. While COPI-coated vesicles accumulated about 12-fold in the presence of GTPγS these vesicles together contained only one fifth of cargo compared to the few vesicles generated in the absence of GTPγS. These data indicate that cargo packaging into COPI-coated vesicles requires hydrolysis of GTP.
Cdc18p can block mitosis by two independent mechanisms
Authors: Greenwood Emma , Nishitani Hideo , Nurse Paul
Published: April 2021

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.
Protein synthesizing units in presynaptic and postsynaptic domains of squid neurons
Authors: Martin Rainer , Vaida Bianca , Bleher Reiner , Crispino Marianna , Giuditta Antonio
Published: April 2021

Putative protein synthesizing domains, called plaques, are characterized in the squid giant synapse and axon and in terminals of squid photoreceptor neurons. Plaques are oval-shaped formations of about 1 μm in size, which (1) generate signals that have spectroscopic electron energy loss characteristics of ribosomes, (2) exhibit ribonuclease-sensitive binding of YOYO-1, a fluorescent RNA/DNA dye, and (3) in part hybridize with a poly(dT) oligonucleotide. In the giant synapse plaques are abundant in the postsynaptic area, but are absent in the presynaptic terminal. In the cortical layer of the optic lobes, plaques are localized in the large carrot-shaped presynaptic terminals of photoreceptor neurons, where they are surrounded by synaptic vesicles and mitochondria.

Biochemical and autoradiographic data have documented that the protein synthetic activity of squid optic lobe synaptosomes is largely due to the presynaptic terminals of the photoreceptor neurons. The identification of ribosomes and poly(A+)-mRNA in the plaques indicates that these structures are sites of local protein synthesis in synaptic domains.
Tau interacts with src-family non-receptor tyrosine kinases
Authors: Lee Gloria , Newman S. Todd , Gard David L. , Band Hamid , Panchamoorthy Govindaswamy
Published: April 2021

Tau and other microtubule-associated proteins promote the assembly and stabilization of neuronal microtubules. While each microtubule-associated protein has distinct properties, their in vivo roles remain largely unknown. Tau is important in neurite outgrowth and axonal development. Recently, we showed that the amino-terminal region of tau, which is not involved in microtubule interactions, is important in NGF induced neurite outgrowth in PC12 cells. Here we report that a proline rich sequence in the amino terminus of tau interacts with the SH3 domains of fyn and src non-receptor tyrosine kinases. Tau and fyn were co-immunoprecipitated from human neuroblastoma cells and co-localization of tau and fyn was visualized in co-transfected NIH3T3 cells. Co-transfection of tau and fyn also resulted in an alteration in NIH3T3 cell morphology, consistent with an in vivo interaction. Fyn-dependent tyrosine phosphorylation of tau occurred in transfected cells and tyrosine phosphorylated tau was identified in human neuroblastoma cells as well. Our data suggest that tau is involved in signal transduction pathways. An interaction between tau and fyn may serve as a mechanism by which extracellular signals influence the spatial distribution of microtubules. The tyrosine phosphorylation of tau by fyn may also have a role in neuropathogenesis, as fyn is upregulated in Alzheimer’s disease.
Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa
Authors: Pu Rongsun , Robinson Kenneth R.
Published: April 2021

The predicted existence of cytoplasmic Ca2+gradients during the photopolarization of the zygotes of the brown algae, Pelvetia and Fucus, has proved to be difficult to establish, and the downstream targets of the putative gradients are not known. We have used quantitative microinjection of the long excitation wavelength Ca2+indicator, Calcium Crimson, and of antibodies against calmodulin to investigate these matters in the zygotes and early embryos of Pelvetia.

We found that there is a window of cytoplasmic Calcium Crimson concentration that gives an adequate signal above autofluorescence yet allows normal development of the zygotes. As Calcium Crimson is not a ratiometric indicator, we injected other zygotes with a Ca2+-insensitive dye, rhodamine B, and imaged the cells at the same time that Calcium Crimson-injected cells were imaged. Ratios were calculated by dividing the averaged pixel values of Calcium Crimson images by the averaged pixel values of corresponding rhodamine B images. By this method, we observed the formation of a cytoplasmic Ca2+ gradient within one hour of the exposure of the cells to unilateral blue light during the photosensitive period. The region of high Ca2+was localized to and predictive of the site of future rhizoid formation. We validated this somewhat indirect method by applying it to the growing rhizoid, where the existence of a tip-localized Ca2+gradient is well established. The method clearly revealed the known gradient.

The injection of ungerminated zygotes with antibodies made against Dictyostelium calmodulin inhibited germination, and this inhibition was abolished if the calmodulin antibodies were coinjected with an excess of purified maize calmodulin. Likewise, the growth of the rhizoids was inhibited by calmodulin antibody injections. The fungus-derived calmodulin antagonist, ophiobolin A, which has previously been shown to be a potent inhibitor of germination, also inhibited rhizoidal growth. Our results provide evidence that a cytoplasmic Ca2+gradient is present during photopolarization and that calmodulin acts as a mediator of Ca2+gradients throughout the early developmental processes of germination and rhizoidal growth in Pelvetia compressa.
Axonal outgrowth of cultured neurons is not limited by growth cone competition
Authors: Lamoureux Phillip , Buxbaum Robert E. , Heidemann Steven R.
Published: April 2021

We have examined the question of scarcity-driven competition for outgrowth among growth cones of a single neuron. We measured spontaneous neurite elongation rates from 85 hours of videotape of the arbors of 31 chick sensory neurons in culture. These rate measurements were analyzed in ten minute periods that allowed cell bodies to be classified as to the number of their growth cones and the elongation to be analyzed as a series of discrete events. Comparing periods in which neurons maintained simple bipolar morphology we find no temporal competition between the two growth cones. That is, periods of above-average growth by one growth cone are not compensated by below-average growth during the same period by its sibling growth cone. Analyzing all outgrowth from a neuron based on its number of growth cones shows that net elongation rate from a single cell body is a linear function of the number of growth cones from 1 to 11. These observations suggest that growth cones behave independently and are not limited by availability of structural precursors. A surplus pool of structural precursors available for normal growth is also indicated by the high capacity for growth from single neurites when experimentally stimulated by mechanical tension. In addition, towing one or more neurites at above average rates does not cause any decline in simultaneous growth cone-mediated outgrowth from a single neuron compared to the 2-3 hour period prior to experimentally induced elongation. This high capacity for growth combined with the often observed, intermittant growth behavior of individual growth cones suggests that neurite outgrowth is intrinsically limited primarily by poor growth cone ‘performance,’ not scarcity-driven competition. We postulate that growth cones are poor ‘tractors,’ exerting too little tension to exploit the available capacity for axonal elongation.
Proteolytic processing and cell biological functions of the amyloid precursor protein
Authors: De Strooper Bart , Annaert Wim
Published: April 2021

Recent research has identified some key players involved in the proteolytic processing of amyloid precursor protein (APP) to amyloid β-peptide, the principal component of the amyloid plaques in Alzheimer patients. Interesting parallels exists with the proteolysis of other proteins involved in cell differentiation, cholesterol homeostasis and stress responses. Since the cytoplasmic domain of APP is anchored to a complex protein network that might function in axonal elongation, dendritic arborisation and neuronal cell migration, the proteolysis of APP might be critically involved in intracellular signalling events.
Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process
Authors: Coverley Dawn , Pelizon Cristina , Trewick Sarah , Laskey Ronald A.
Published: April 2021

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G1 nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G1 phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6.

Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G2 then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G1 phase.
Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas
Authors: Finst Rip J. , Kim Peter J. , Griffis Eric R. , Quarmby Lynne M.
Published: April 2021

A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca2+-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca2+-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca2+/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca2+-activated, Ca2+-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.
Genetic defects in acetylcholine signalling promote protein degradation in muscle cells of Caenorhabditis elegans
Authors: Szewczyk Nathaniel J. , Hartman James J. , Barmada Sami J. , Jacobson Lewis A.
Published: April 2021

A myosin-lacZ fusion, expressed in 103 muscle cells of Caenorhabditis elegans, reports on how proteolysis in muscle is controlled by neural and intramuscular signals. Upon acute starvation, the fusion protein is degraded in the posterior 63 cells of the body-wall muscle, but remains stable in 32 anterior body-wall muscles and 8 vulval muscle cells. This distinction correlates with differences in the innervation of these cells. Reporter protein in the head and vulval muscles becomes labile upon genetic ‘denervation’ in mutants that have blocks in pre-synaptic synthesis or release of acetylcholine (ACh) or post-synaptic reception at nicotinic ACh receptors (nAChR), whereas protein in all 103 muscles is stabilized by the nicotinic agonist levamisole in the absence of ACh production. Levamisole does not stabilize muscle protein in nAChR mutants that are behaviorally resistant to levamisole. Neural inputs thus exert negative control over the proteolytic process in muscle by stimulating muscle nicotinic ACh receptors.
The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2)
Authors: Litman Thomas , Brangi Mariafiorella , Hudson Eric , Fetsch Patricia , Abati Andrea , Ross Douglas D. , Miyake Keisuke , Resau James H. , Bates Susan E.
Published: April 2021

Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.
The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia
Authors: Chew Catherine S. , Parente John A. , Chen Xunsheng , Chaponnier Christine , Cameron Richard S.
Published: April 2021

Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the γ-isoform of actin in unstimulated cells, to the β-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.
Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing
Authors: Arias Ariel E. , Vélez-Granell Carlos S. , Mayer Gaétan , Bendayan Moïse
Published: April 2021

Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin- secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.
Retroviral expression of Wnt-1 and Wnt-7b produces different effects in mouse mammary epithelium
Authors: Naylor Stuart , Smalley Matthew J. , Robertson David , Gusterson Barry A. , Edwards Paul A. W. , Dale Trevor C.
Published: April 2021

Several Wnt genes are expressed in the postnatal mouse mammary gland and are thought to be involved in mammary gland development. Ectopic expression of Wnt-1, which is not normally expressed in the mammary gland, drives the formation of a pre-neoplastic hyperplasia. Cell culture-based assays have shown that Wnt-1 and some mammary-expressed Wnts transform C57MG cells. This has led to the suggestion that Wnt-1 functions as an oncogene through the inappropriate activation of developmental events that are normally controlled by the ‘transforming’ class of Wnts. In this study, Wnt-7b was expressed in vivo using recombinant retroviruses. Wnt-7b did not alter normal mammary gland development despite having similar effects to Wnt-1 in cell culture. We conclude that the in vitro classification of Wnts as ‘transforming’ does not correlate with the transformation in vivo. To facilitate the analysis of Wnt-expression, a lacZ-containing, bicistronic recombinant retrovirus was developed. Immunohistochemistry and electron microscopy identified retrovirally transduced myoepithelial and luminal epithelial cells in normal and hyperplastic tissues. The distribution of transduced cells in mammary outgrowths was consistent with current models of mammary stem cell identity.
Prostanoids mediate the protective effect of trefoil factor 3 in oxidant-induced intestinal epithelial cell injury: role of cyclooxygenase-2
Authors: Tan Xiao-Di , Chen Yi-Hua , Liu Qian-Ping , Gonzalez-Crussi Frank , Liu Xue-Li
Published: April 2021

Trefoil factors are small peptides found in several mammalian tissues including gut, respiratory tract and brain. Their physiological function is not well understood. Among them, trefoil factor 3 (intestinal trefoil factor) is known to be cytoprotective in the gut. However, the molecular mechanism and secondary mediators of trefoil factor 3 action are not known. In the present study, we examined whether the cyclooxygenase pathway is involved in trefoil factor 3 action. We showed that trefoil factor 3 significantly induces the production of prostaglandin E2 and prostaglandin I2 in IEC-18 cells (an intestinal epithelial cell line) in a dose dependent manner. Western blot and immunohistochemistry revealed that trefoil factor 3 (2.5 μM) up-regulates the expression of cyclooxygenase-2 but not cyclooxygenase-1 in IEC-18 cells. Treating cells with trefoil factor 3 (10 μM) significantly attenuated reactive oxygen species-induced IEC-18 cell injury. This effect is blocked by NS-398 (10 μM), a selective cyclooxygenase-2 inhibitor. Moreover, we demonstrated that exogenously administered carbacyclin (1 μM, a stable analogue of prostaglandin I2) and/or prostaglandin E2 (1 μM) caused a significant reduction of reactive oxygen species-induced cell injury, mimicking the effect of trefoil factor 3. In summary, our results indicate that trefoil factor 3 activates cyclooxygenase-2 in intestinal epithelium to produce prostaglandin I2 and prostaglandin E2, which function as survival factors and mediate the cytoprotective action of trefoil factor 3 against oxidant injury.
Relative locations of the centromere and imprinted SNRPN gene within chromosome 15 territories during the cell cycle in HL60 cells
Authors: Nogami Masahiro , Kohda Atsushi , Taguchi Hiroshi , Nakao Mitsuyoshi , Ikemura Toshimichi , Okumura Katsuzumi
Published: April 2021

Investigations of imprinted regions provide clues that increase our understanding of the regulation of gene functions at higher order chromosomal domains. Here, the relative positions of the chromosome 15 centromere and the imprinted SNRPN gene in interphase nuclei of human myeloid leukemia HL60 cells were compared, because the homologous association of this imprinted chromosomal domain was previously observed in lymphocytes and lymphoblasts. Four targets including the chromosome 15 territory, its centromere, the SNRPN gene on this chromosome, and the nucleus, were visualized simultaneously in three-dimensionally preserved nuclei using multicolor fluorescence in situ hybridization, and the spatial distributions of these probes were analyzed with a cooled CCD camera deconvolution system. We found that preferential association of SNRPN interhomologues did not occur during the cell cycle in HL60 cells, although this gene exhibited asynchronous replication and monoallelic expression in this cells. SNRPN was found to localize at the periphery of the chromosome territories, and it preferentially faced the nuclear membrane, unlike the adjacent centromeric repeat. The SNRPN gene and the centromere were located close to each other late in S phase, reflecting that these DNA segments may be compacted into the same intranuclear subcompartments with the progress of S phase and in course of preparation for the following G2 phase. Our results suggest that, although an imprinted gene has features similar to those observed with intranuclear localization of other gene coding sequences, the characteristic of mutual recognition of imprinted regions is determined by certain cellular regulation, and it is not necessary for the allele-specific features of an imprinted gene.
A submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells
Authors: Schmidt Katja , Dartsch Heidrun , Linder Dietmar , Kern Horst-Franz , Kleene Ralf
Published: April 2021

The secretory lectin ZG16p mediated the binding of aggregated zymogens to the granule membrane in pancreatic acinar cells. Using a recently established in vitro condensation-sorting assay, we now show that pretreatment of zymogen granule membranes (ZGM) with either sodium bicarbonate at pH 10 or with phosphatidyl inositol-specific phospholipase C (PI-PLC) reduced the binding efficiency of zymogens to the same extent, as distinct components were liberated from ZGM. Analysis of the composition of the bicarbonate extract revealed the presence of the secretory lectin ZG16p, the serpin ZG46p and the GPI-linked glycoprotein GP-2, together with several unknown proteins, and small amounts of lipase and carboxylester lipase. The unknown proteins detected in 2-D gels represented a group of acidic and basic protein spots, which were positive in a glycan staining reaction and were soluble in methanol. One protein spot of the acidic group and several of the basic group reacted with a monoclonal antibody directed against chondroitin sulfate, indicating that the proteins represented proteoglycans. A staining pattern similar to the glycan reaction was observed in immunoblots using a polyclonal antibody directed against the whole bicarbonate extract. Immunogold electron microscopy revealed that this antibody reacted with components in the periphery of zymogen granules and strongly stained ZGM in the pellet fraction of a standard in vitro condensation-sorting assay. The amino acid composition of isolated components of both the acidic and basic group showed similarities to aggrecan, a cartilage-specific proteoglycan, and to glycine-rich glycoproteins, respectively. We therefore conclude that a submembranous matrix on the ZGM composed of proteoglycans and glycoproteins is involved in granule formation in pancreatic acinar cells.
An upstream element of the TamS1 gene is a site of DNA-protein interactions during differentiation to the merozoite in Theileria annulata
Authors: Shiels Brian , Fox Mark , McKellar Sue , Kinnaird Jane , Swan David
Published: April 2021

Apicomplexan parasites are major pathogens of humans and domesticated animals. A fundamental aspect of apicomplexan biology, which may provide novel molecular targets for parasite control, is the regulation of stage differentiation. Studies carried out on Theileria annulata, a bovine apicomplexan parasite, have provided evidence that a stochastic process controls differentiation from the macroschizont to the merozoite stage. It was postulated that this process involves the presence of regulators of merozoite gene expression in the preceding stage of the life cycle, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run-on analysis showed that TamS1 expression is controlled, at least in part, at the transcriptional level. The transcription start site showed homology with the consensus eukaryotic initiator motif, and study of the 5′ upstream region by the electrophoretic mobility-shift assay demonstrated that a 23 bp motif specifically bound factors from parasite-enriched nuclear extracts. Three complexes were shown to bind to a 9 bp core binding site (5′-TTTGTAGGG-3′). Two of these complexes were present in macroschizont extracts but were found at elevated levels during differentiation. Both complexes contain a polypeptide of the same molecular mass and may be related via the formation of homodimer or heterodimer complexes. The third complex appears to be distinct and was detected at time points associated with the transition to high level merozoite gene expression.
The central matrix loop drives import of uncoupling protein 1 into mitochondria
Authors: Schleiff Enrico , McBride Heidi
Published: April 2021

The uncoupling protein (UCP1) is a carrier protein of the inner mitochondrial membrane spanning the bilayer six times. It does not contain a typical amino-terminal targeting signal and the mechanism of targeting and insertion is unknown. Here we focus on the biogenesis of UCP1 by analysing the import signals contained within the three repeated units of the protein. The amino-terminal third of the protein can mediate insertion into the outer membrane and therefore acts as artificial targeting signal when fused to DHFR. However, in the context of full-length UCP, the targeting information contained within the first repeated unit is not sufficient to trigger insertion into the outer membrane. Deletion of either the first or third repeated unit from UCP1 did not reduce import into the inner membrane and bound to the outer membrane receptor protein hTom20 with the characteristics of full-length UCP1. Deletion of the second repeat of UCP1 completely abolished all import into the mitochondria. Consistent with this, the central repeat alone was efficiently imported to the inner membrane and bound hTom20 with the characteristics of UCP1. We conclude that the site for binding hTom20 is within the central repeat and that this domain contains the complete targeting signal for directing UCP1 to the inner membrane.
Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA
Authors: Calado Angelo , Carmo-Fonseca Maria
Published: April 2021

The nuclei of mammalian cells contain domains, termed nuclear speckles, which are enriched in splicing factors and poly(A) RNA. Although nuclear speckles are thought to represent reservoirs from which splicing factors are recruited to sites of transcription and splicing, the presence of poly(A) RNA in these structures remains enigmatic. An additional component of the speckles is poly(A) binding protein 2 (PABP2), a protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we investigated whether PABP2 contributes to the targeting of poly(A) RNA to the speckles. The results show that localization of PABP2 in speckles is independent of import of the protein into the nucleus. Inhibition of transcription or poly(A) synthesis at the end of mitosis does not affect nuclear import of PABP2 but prevents its localization to speckles. Furthermore, PABP2 mutants with decreased ability to bind to poly(A) fail to localize to speckles. Taken together the results show that PABP2 localizes to the nuclear speckles as a consequence of its binding to poly(A) RNA, contrasting to splicing factors which assemble into speckles in the absence of newly synthesized transcripts.
Roles of phospholipid signaling in chemoattractant-induced responses
Authors: Wu Dianqing , Huang Chi-Kuang , Jiang Huiping
Published: April 2021

Chemoattractants, including chemokines, play a central role in regulation of inflammatory reactions by attracting and activating leukocytes. These molecules have been found to regulate metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) via phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Recent studies of mouse lines that lack PLC-β2, PLC-β3, or PI3Kγ demonstrate that chemoattractants act through PLC-β2 and PLC-β3 to hydrolyze PtdIns(4,5)P2 and through PI3Kγ to phosphorylate PtdIns(4,5)P2 in mouse neutrophils. These studies also confirmed the importance and revealed new roles of these signaling pathways in chemoattractant-induced responses.
The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor
Authors: Poukka Hetti , Karvonen Ulla , Yoshikawa Noritada , Tanaka Hirotoshi , Palvimo Jorma J. , Jänne Olli A.
Published: April 2021

The androgen receptor (AR) is a transcription factor that mediates androgen action. We have used the green fluorescent protein (GFP) technique to investigate dynamics of nuclear trafficking of human AR in living cells. In the absence of ligand, the GFP-AR fusion protein is distributed between cytoplasm and nuclei. Androgen exposure leads to a rapid and complete import of GFP-AR to nuclei of CV-1 cells (≥90% nuclear in 30 minutes), whereas a pure antiandrogen, Casodex, elicits a slower (<40% nuclear in 30 minutes) and incomplete transfer. Unliganded ARs with mutations in the basic amino acids of the bipartite nuclear localization signal (NLS) within the second zinc finger and the hinge region are predominantly cytoplasmic and their androgen-dependent nuclear import is severely compromised (≤20% nuclear in 30 minutes). Interestingly, substitutions of the Leu residues flanking the bipartite NLS lead to inefficient nuclear transfer in response to androgen (≤20% nuclear in 30 minutes). The ligand-binding domain of AR, which represses bipartite NLS activity, contains an agonist-specific NLS. The small nuclear RING finger protein SNURF, which interacts with AR through a region overlapping with the bipartite NLS, facilitates AR import to nuclei and retards its export on hormone withdrawal. More AR is associated with the nuclear matrix in the presence than absence of coexpressed SNURF. We suggest that the SNURF-mediated tethering of AR in nuclei represents a novel mechanism for activating steroid receptor functions.
Activation of protein kinase Cα inhibits growth of pancreatic cancer cells via p21cip-mediated G1 arrest
Authors: Detjen K. M. , Brembeck F. H. , Welzel M. , Kaiser A. , Haller H. , Wiedenmann B. , Rosewicz S.
Published: April 2021

We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)α. PKCα subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKCα activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCα, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKCα activation arrested cells in the G1 phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21cip1 cyclin-dependent kinase inhibitors. Levels of p21cip1 remained elevated and CDK2 activity repressed in spite of PKCα downregulation, indicating that downstream effectors of PKCα are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCα-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCα results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.
Pyk2 and FAK differentially regulate progression of the cell cycle
Authors: Zhao Jihe , Zhen Chuanhai , Guan Jun-Lin
Published: April 2021

We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G1 to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant ΔC14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.
Drosophila D-Titin is required for myoblast fusion and skeletal muscle striation
Authors: Zhang Yong , Featherstone David , Davis Warren , Rushton Emma , Broadie Kendal
Published: April 2021

An ethylmethane sulfonate (EMS) mutagenesis of Drosophila melanogaster aimed at discovering novel genes essential for neuromuscular development identified six embryonic lethal alleles of one genetic locus on the third chromosome at 62C. Two additional lethal P element insertion lines, l(3)S02001 and l(3)j1D7, failed to complement each other and each of the six EMS alleles. Analysis of genomic sequence bracketing the two insertion sites predicted a protein of 16,215 amino acid residues, encoded by a 70 kb genomic region. This sequence includes the recently characterized kettin, and includes all known partial D-Titin sequences. We call the genetic locus, which encodes both D-Titin and kettin, D-Titin. D-Titin has 53 repeats of the immunoglobulin C2 domain, 6 repeats of the fibronectin type III domain and two large PEVK domains.

Kettin appears to be the NH2-terminal one third of D-Titin, presumably expressed via alternative splicing. Phenotype assays on the allelic series of D-Titin mutants demonstrated that D-Titin plays an essential role in muscle development. First, D-Titin has an unsuspected function in myoblast fusion during myogenesis and, second, D-Titin later serves to organize myofilaments into the highly ordered arrays underlying skeletal muscle striation. We propose that D-Titin is instrumental in the development of the two defining features of striated muscle: the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.
Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network
Authors: Blum Robert , Stephens David J. , Schulz Irene
Published: April 2021

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 μm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo.

Movies available on-line (http://www.biologists.com/JCS/movies/jcs1334.html), Movie ADDITION, Movie FIGURE 2, Movie FIGURE 3, Movie FIGURE 4, Movie FRAP, Movie GFP_P23, Movie OVERVIEW, Movie POST_GOL
Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of α5β1 integrin expression
Authors: Duncan Melinda K. , Kozmik Zbynek , Cveklova Kveta , Piatigorsky Joram , Cvekl Ales
Published: April 2021

The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120ctn as well as large aggregates of α5β1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human α5- and β1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human α5 and β1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.