bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of MYO4, a second class V myosin gene in yeast
Authors: Haarer B. K. , Petzold A. , Lillie S. H. , Brown S. S. 
Published: April 2021

ABSTRACT
We have isolated a fourth myosin gene (MYO4) in yeast (Saccharomyces cerevisiae). MYO4 encodes a ∼170 kDa (1471 amino acid) class V myosin, using the classification devised by Cheney et al. (1993a; Cell Motil. Cytoskel. 24, 215-223); the motor domain is followed by a neck region containing six putative calmodulin-binding sites and a tail with a short potential ‘coiled-coil’ domain. A comparison with other myosins in GenBank reveals that Myo4 protein is most closely related to the yeast Myo2 protein, another class V myosin. Deletion of MYO4 produces no detectable phenotype, either alone or in conjunction with mutations in myo2 or other myosin genes, the actin gene, or secretory genes. However, overexpression of MYO4 or MYO2 results in several morphological abnormalities, including the formation of short strings of unseparated cells in diploid strains, or clusters of cells in haploid strains. Alterations of MYO4 or MYO2 indicate that neither the motor domains nor tails of these myosins are required to confer the over-expression phenotype, whereas the neck region may be required. Although this phenotype is similar to that seen upon MYO1 deletion, we provide evidence that the overexpression of Myo4p or Myo2p is not simply interfering with Myo1p function.
Determinants of Drosophila zw10 protein localization and function
Authors: Williams Byron C. , Goldberg Michael L. 
Published: April 2021

ABSTRACT
We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.
Intercellular communication between epithelial and fiber cells of the eye lens
Authors: Bassnett Steven , Kuszak Jerome R. , Reinisch Lou , Brown Henry G. , Beebe David C. 
Published: April 2021

ABSTRACT
Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit ‘metabolic cooperation’ between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4°C or by treatment with octanol and which excluded large (∼10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.
The dynein genes of paramecium tetraurelia: sequences adjacent to the catalytic p-loop identify cytoplasmic and axonemal heavy chain isoforms
Authors: Asai David J. , Beckwith Susan M. , Kandl Kimberly A. , Keating Heather H. , Tjandra Hendri , Forney James D. 
Published: April 2021

ABSTRACT
Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary β heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein. Indirect immunofluorescence microscopy showed that the motif B epitope was excluded from cilia. These results suggest that sequence motif A may be present in both classes of dynein but that sequence motif B is restricted to cytoplasmic dynein isoforms. This study provides direct evidence that individual dynein gene products are spatially segregated in a single cell.
Nuclear export of signal recognition particle RNA is a facilitated process that involves the Alu sequence domain
Authors: He Xiao-Ping , Bataillé Nelly , Fried Howard M. 
Published: April 2021

ABSTRACT
The signal recognition particle is a cytoplasmic RNA-protein complex that mediates translocation of secretory polypeptides into the endoplasmic reticulum. We have used a Xenopus oocyte microinjection assay to determine how signal recognition particle (SRP) RNA is exported from the nucleus. Following nuclear injection, SRP RNA accumulated in the cytoplasm while cytoplasmically injected SRP RNA did not enter the nucleus. Cytoplasmic accumulation of SRP RNA was an apparently facilitated process dependent on limiting trans-acting factors, since nuclear export exhibited saturation kinetics and was completely blocked either at low temperature or by wheat germ agglutinin, a known inhibitor of nuclear pore-mediated transport. At least one target for trans-acting factors that promote nuclear export of SRP RNA appears to be the Alu element of the molecule, since a transcript consisting of only the Alu sequence was exported from the nucleus in a temperature-dependent manner and the Alu transcript competed in the nucleus for transport with intact SRP RNA. Although the identities of trans-acting factors responsible for SRP RNA transport are at present unknown, we suggest that proteins contained within the cytoplasmic form of SRP are candidates. Consistent with this idea were the effects of a mutation in SRP RNA that prevented binding of two known SRP proteins to the Alu sequence.
A chimera of the cytoplasmic tail of the mannose 6-phosphate/igf-ii receptor and lysozyme localizes to the tgn rather than prelysosomes where the bulk of the endogenous receptor is found
Authors: Conibear E. , Pearse B. M. F. 
Published: April 2021

ABSTRACT
We fused the cytoplasmic and transmembrane domains of the bovine mannose 6-phosphate/IGF-II receptor (MPR) to lysozyme, a monomeric secretory protein thought to be devoid of sorting information. When the resulting chimera (lys/MPR) was transiently expressed in COS cells or stably expressed in CV1 cells, it had a predominantly intracellular distribution in the trans-Golgi region, with less than 10% present on the surface. In contrast, a similar chimera containing the transmembrane and cytoplasmic domains of the low density lipoprotein receptor (lys/LDLR) was localized to the plasma membrane, even though it endocytoses efficiently. Exchanging domains between the lys/MPR and lys/LDLR chimeras indicated that the MPR cytoplasmic domain contains the information necessary to specify the intracellular localization of the chimeric molecule. This signal must be located in the membrane-proximal third of the tail, as deletion of the last 120 residues of the 163 residue tail has no obvious effect on the distribution of lys/MPR.
However, the recycling of the lys/MPR does not completely mimic that of the intact endogenous MPR, as immunofluorescence labelling shows that they are predominantly in different locations, indicating a role for the lumenal domain of the MPR in determining the steady-state distribution of the MPR itself.
Expression of chimeric connexins reveals new properties of the formation and gating behavior of gap junction channels
Authors: Bruzzone Roberto , White Thomas W. , Paul David L. 
Published: April 2021

ABSTRACT
Direct intercellular communication occurs through specialized channels, which are formed by the interaction of two half-channels, or connexons, contributed by each of the two participating cells. The ability to establish intercellular communication is specified, in part, by the expression of different structural proteins, termed connexins. Connexins can control the establishment of intercellular communication by selectively pairing with some but not other family members. To characterize the protein domains that allow connexins to recognize and discriminate between alternative partners, we have created chimeras composed of selected regions of rat connexin43, which forms channels with Xenopus connexin38, and rat connexin32, which cannot. Pairs of Xenopus oocytes were used to test the ability of the chimeras to form homotypic channels with themselves, and heterotypic channels with the parent connexins or with endogenous Xenopus connexin38. While all hybrid molecules tested were efficiently expressed by oocytes, most were devoid of functional activity. A chimera consisting of connexin32 from the N terminus to the second transmembrane domain, fused to connexin43 from the middle cytoplasmic loop to the C terminus, designated as 3243H4, was able to pair functionally with Xenopus connexin38 and one of its parent connexins, connexin43. Voltage-dependent closure of heterotypic channels containing 3243H4 was asymmetric, exhibited novel characteristics that were not predicted by the behavior of the parent connexins and was dependent on the type of connexin with which 3243H4 was paired. In contrast, 3243H4 was unable to form functional channels with either itself or the other parent, connexin32. Together, these results suggest that these connexins are not composed of functionally exchangeable regions and that multiple domains, namely the middle cytoplasmic portion and the second extracellular domain, can influence the interactions between connexins present in adjacent cells. Furthermore, they indicate that voltage gating is not strictly intrinsic behavior for a given connexin, but can be modulated by the partner connexins to which they are paired. Finally, the finding that 3243H4 is functional only in heterotypic configurations, and cannot form homotypic channels, suggests the existence of a novel form of selectivity: self-discrimination. The latter property may represent another mechanism that operates to control the extent of communication between cells.
Different effects of protein kinase inhibitors on the localization of junctional proteins at cell-cell contact sites
Authors: Denisenko Natasha , Burighel Paolo , Citi Sandra 
Published: April 2021

ABSTRACT
The protein kinase inhibitor H-7 prevents the assembly of tight junctions in cultured Madin Darby Canine Kidney (MDCK) epithelial cells (Balda et al. (1991)J. Membr. Biol. 122, 193-202; Nigam et al. (1991)Biochem. Biophys. Res. Commun. 181, 548-553); however, its mechanism of action is unknown. To understand the basis of the activity of H-7 and other inhibitors we compared the effect of H-7 on the localization of proteins belonging to tight junctions and adherens-type junctions (zonula adhaerens and desmosome), and on the organization of actin microfilaments. Junction assembly was induced in MDCK cells either by the ‘Ca2+ switch’ procedure or by incubating trypsinized cells at normal extracellular Ca2+, and the cells were then immunofluorescently labeled with antibodies against cingulin, ZO-1, E-cadherin and desmoplakin, and with FITC-phalloidin. Here we show by measuring the transepithelial resistance that, in addition to H-7, H-8 and staurosporine can also significantly block the assembly of tight junctions, whereas HA1004 is poorly active. H-7 inhibited the accumulation of cingulin and ZO-1 in junctional areas most effectively when added during assembly at normal extracellular Ca2+. On the other hand, H-7 did not have major effects on the accumulation of E-cadherin and desmoplakin in the regions of cell-cell contact using either assembly protocol. Electron microscopy confirmed that H-7 does not abolish the formation of adherens-type junctions, suggesting that phosphorylation plays a different role in the assembly of tight junctions versus adherens-type junctions. Finally, in both protocols of junction assembly H-7 caused a major disorganization of actin microfilaments, suggesting that H-7 may prevent TJ assembly through its effect on the cytoskeleton.
E-cadherin is the major mediator of human melanocyte adhesion to keratinocytes in vitro
Authors: Tang Aimin , Eller Mark S. , Hara Masahiro , Yaar Mina , Hirohashi Setsuo , Gilchrest Barbara A. 
Published: April 2021

ABSTRACT
E- and P-cadherin are calcium (Ca2+)-dependent cell adhesion molecules important in the morphogenesis and maintenance of skin structure. By use of flow cytometry and specific antibodies, we now show that cultured human melanocytes express E- and P-cadherin on their surfaces, and that these molecules have the same characteristics as reported for other cell types. Specifically, melanocyte cadherins are sensitive to trypsin digestion in the absence of Ca2+ and are protected from trypsin degradation by Ca2+, and are functional at 37°C but not at 4°C. We further show that melanocytes contain mRNA transcripts encoding both E- and P-cadherin. Adhesion of cultured melanocytes to keratinocyte monolayers is abolished by pre-treatment of the melanocytes with trypsin/EDTA, which degrades E- and P-cadherins, is greatly reduced by anti-E-cadherin antibodies and is slightly reduced by antibodies to P-cadherin, α2, α3 and β1 integrins. In contrast to normal melanocytes, eight of nine melanoma cell lines lacked E-cadherin (or expressed markedly reduced levels) and five were negative for P-cadherin. Melanoma cells also failed to adhere to keratinocyte monolayers. These results demonstrate that normal human melanocytes express functional E- and P-cadherin and that E-cadherin is primarily responsible for adhesion of human melanocytes to keratinocytes in vitro. In addition, transformed melanocytes express markedly reduced levels of E- and P-cadherin, and exhibit decreased affinity for normal keratinocytes in vitro, suggesting that loss of cadherins may play a role in melanoma metastasis.
Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells
Authors: Asada Tetsuhiro , Shibaoka Hiroh 
Published: April 2021

ABSTRACT
As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubuledependent ATPase and GTPase activity caused minus-endheaded gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 μm/minute in the presence of ATP and 0.50 pm/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.
Dermal fibroblasts activate keratinocyte outgrowth on collagen gels
Authors: Tuan Tai-Lan , Keller Lia C. , Sun David , Nimni Marcel E. , Cheung David 
Published: April 2021

ABSTRACT
The effects of dermal fibroblasts on keratinocyte outgrowth on collagen substrata was studied using an in vitro ker-atinocyte-collagen gel composite model. Skin fibroblasts were seeded inside collagen gels, which remained attached to the cell culture plastic substratum. Fibroblasts incorporated in collagen gels were either kept viable throughout the study, or were lysed hypotonically with water at different time intervals (2 hours and 5 days). Results show that very little keratinocyte outgrowth occurred on either plain collagen gels or gels that had previously contained viable fibroblasts for 2 hours. A 3-to 4-fold increase in keratinocyte outgrowth occurred on collagen gels that had previously contained viable fibroblasts for 5 days. A striking increase (20-fold) in keratinocyte outgrowth was observed on collagen gels that contain viable fibroblasts. The effect of fibroblast diffusible factors on keratinocyte outgrowth was further studied with a co-culture system using Millicell inserts. It was found that the co-culture of fibroblasts with the composite enhanced keratinocyte outgrowth on collagen gels that had previously contained viable fibroblasts for 5 days. Among all, however, the keratinocyte outgrowth was far better on gels containing viable fibroblasts. Addition of keratinocyte growth factor or its neutralizing antibody did not affect keratinocyte outgrowth. These results suggest that dermal fibroblasts can activate keratinocyte outgrowth on collagen matrices through some diffusible factors other than keratinocyte growth factor, and epithelial-mesenchymal interactions exert some special effects on keratinocyte outgrowth on collagen gels.
Developmental regulation of polyglutamylated α- and β-tubulin in mouse brain neurons
Authors: Audebert Stéphane , Koulakoff Annette , Berwald-Netter Yoheved , Gros François , Denoulet Philippe , Eddé Bernard 
Published: April 2021

ABSTRACT
Polyglutamylation is an important posttranslational modi-fication of tubulin that is very active in nerve cells, where it accounts for the main factor responsible for tubulin het-erogeneity. In the present work, we have analyzed quantitative and qualitative changes in glutamylated α- and β-tubulin occurring during neuronal differentiation in culture. Glutamylated α- and β-tubulin both markedly accumulate during this process with a time course remark-ably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation of the two subunits are not exactly the same. Glutamylated α-tubulin is already abundant in very young neurons and displays, at this stage, a wide range of its degree of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated β-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into α- and β-tubulin decreases during differentiation, as well as the rate of the reverse deglu-tamylation reaction, suggesting that accumulation of glu-tamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin.
Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable α-tubulin, which can occur in combination with polyglu-tamylation and contributes to increase the complexity of tubulin in mature neurons.