bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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.
SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes
Authors: Alkemade J. A. C. , Molhuizen H. O. F. , Ponec M. , Kempenaar J. A. , Zeeuwen P. L. J. M. , Jongh G. J. de , Vlijmen-Willems I. M. J. J. van , Erp P. E. J. van , Kerkhof P. C. M. van de , Schalkwijk J.
Published: April 2021

Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8–10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.

These findings demonstrate that SKALP is an inducible proteinase inhibitor and support the concept that SKALP acts as a regulatory molecule in cutaneous homeostasis.
Characterization of very acidic phagosomes in breast cancer cells and their association with invasion
Authors: Montcourrier P. , Mangeat P. H. , Valembois C. , Salazar G. , Sahuquet A. , Duperray C. , Rochefort H.
Published: April 2021

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 μm) in which endocy-tosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcour-rier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epiflu-orescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 μm diameter latex beads and fluorescent Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are het-erophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H+-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (<4), lower than the lysosomal value (pH ∼5) in the same cells and in specialized phagocytotic cells such as macrophages.

We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.
Control of stable lamellipodia formation by expression of cell surface β1,4-galactosyltransferase cytoplasmic domains
Authors: Appeddu Paul A. , Shur Barry D.
Published: April 2021

Mesenchymal cell migration on basal lamina is mediated, in part, by the binding of cell surface β1,4-galactosyltrans-ferase (GalTase) to specific N-linked oligosaccharides in the E8 domain of laminin. On migrating cells, surface GalTase is anchored to the cytoskeleton; when GalTase is prevented from associating with the cytoskeleton, lamellipodia formation and subsequent migration are inhibited. To define better the involvement of GalTase-cytoskeleton interactions in cell motility, we examined the lamellipodia formation, polarity and migratory behavior of stably trans-fected 3T3 fibroblasts expressing increased or decreased levels of GalTase capable of interacting with the cytoskele-ton. Initially, the motile behavior of individual cells was quantified in the absence of exogenous stimuli. Cells that overexpress GalTase binding sites for the cytoskeleton changed their polarity more frequently and translocated more erratically than did control cells when assayed on laminin substrata. These differences were not observed, however, when cells were plated on fibronectin, which does not contain binding sites for surface GalTase. GalTase-transfected cells were also assayed for their ability to polarize in response to a specific stimulus. In this case, the ability of a cell to reorient towards a gradient of platelet-derived growth factor was found to be directly proportional to the amount of GalTase associated with the cytoskeleton. Differences in response to platelet-derived growth factor were not due to differences in growth factor binding. Indirect immunofluorescence showed that altering the level of GalTase did not affect the ventrally distributed pool of GalTase stably associated with the cytoskeleton; however, stress fiber formation was inhibited. Thus, increasing surface GalTase binding sites for the cytoskele-ton leads to erratic, multipolar behavior in the absence of any vectorial stimulus, but the ability to form a functional lamellipodium in response to a stimulus is dependent upon the amount of surface GalTase associated with the cytoskeleton. Apparently, cells are able to regulate cytoskeletal assembly and lamellipodial stability by altering the expression and/or affinity of appropriate matrix receptors, such as GalTase, and their correspond-ing binding sites in the cytoskeleton.
The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum
Authors: Watson Ned , McGuire Vince , Alexander Stephen
Published: April 2021

The PsB glycoprotein in Dictyostelium discoideum is one of a diverse group of developmentally regulated, prespore-cell-specific proteins, that contain a common O-linked oligosaccharide. This post-translational modification is dependent on the wild-type modB allele. The PsB protein exists as part of a multiprotein complex of six different proteins, which have different post-translational modifications and are held together by both covalent and noncovalent interactions (Watson et al. (1993). J. Biol. Chem. 268, 22634-22641). In this study we have used microscopic and biochemical analyses to examine the cellular localization and function of the PsB complex during development. We found that the PsB complex first accumulates in prespore vesicles in slug cells and is secreted later during culmination and becomes localized to both the extracellular matrix of the apical spore mass of mature fruiting bodies and to the inner layer of the spore coat. The PsB associated with the spore coat is covalently bound by disulfide bridges. The PsB protein always exists in a multiprotein complex, but the composition of the PsB complex changes during secretion and spore maturation. Some of the PsB complex proteins have been identified as spore coat proteins. These data demonstrate that some of the proteins that form the spore coat exist as a preassembled precursor complex. The PsB complex is secreted in a developmentally regulated manner during the process of spore differentiation, at which time proteins of the complex, as well as additional spore coat proteins, become covalently associated in at least two forms of extracellular matrix: the interspore matrix and the spore coat. These and other studies show that proteins with modB dependent O-linked oligosaccharides are involved in a wide variety of processes underlying morphogenesis in this organism. These developmental processes are the direct result of cellular mechanisms regulating protein targeting, assembly and secretion, and the assembly of specific extracellular matrices.
Elastin exhibits a distinctive temporal and spatial pattern of distribution in the developing chick limb in association with the establishment of the cartilaginous skeleton
Authors: Hurle Juan M. , Corson Glen , Daniels Karla , Reiter Rebecca S. , Sakai Lynn Y. , Solursh Michael
Published: April 2021

In this work we have analyzed the presence of elastic components in the extracellular matrices of the developing chick leg bud. The distributions of elastin and fibrillin were studied immunohistochemically in whole-mount preparations using confocal laser microscopy. The association of these constituents of the elastic matrix with other components of the extracellular matrix was also studied, using several additional antibodies. Our results reveal the transient presence of an elastin-rich scaffold of extracellular matrix fibrillar material in association with the establishment of the cartilaginous skeleton of the leg bud. The scaffold consisted of elastin-positive fibers extending from the ectodermal surface of the limb to the central cartilageforming regions and between adjacent cartilages. Fibrillin immunolabeling was negative in this fibrillar scaffold while other components of the extracellular matrix including: tenascin, laminin and collagens type I, type III and type VI; appeared codistributed with elastin in some regions of the scaffold. Progressive changes in the spatial pattern of distribution of the elastin-postive scaffold were detected in explant cultures in which one expects a modification in the mechanical stresses of the tissues related to growth.

A scaffold of elastin comparable to that found in vivo was also observed in high-density micromass cultures of isolated limb mesodermal cells. In this case the elastic fibers are observed filling the spaces located between the cartilaginous nodules. The fibers become reoriented and attach to the ectodermal basal surface when an ectodermal fragment is located at the top of the growing micromass. Our results suggest that the formation of the cartilaginous skeleton of the limb involves the segregation of the undifferentiated limb mesenchyme into chondrogenic and elastogenic cell lineages. Further, a role for the elastic fiber scaffold in coordinating the size and the spatial location of the cartilaginous skeletal elements within the limb bud is also suggested from our observations.
Nucleolar localisation of three Hox homeoproteins
Authors: Corsetti Maria Teresa , Levi Giovanni , Lancia Flavia , Sanseverino Lorenza , Ferrini Silvano , Boncinelli Edoardo , Corte Giorgio
Published: April 2021

Homeoproteins encoded by genes of the Hox family are nuclear proteins believed to act as transcription factors and to participate in the determination of the body plan. Here we show that in several vertebrate cells, they exhibit a sub-nuclear localisation associated with the nucleolus.

We used monoclonal antibodies to study the distribution of three homeoproteins, namely HOXB7, HOXC6 and HOXD4. The immunoreactivity to antibodies against HOXC6 protein in Xenopus laevis embryonic tissues is restricted to one or two spots within the nucleus; this dis-tribution partially overlaps that of fibrillarin, a protein of the fibrillar zone of the nucleoli. Indirect immunofluorescence analysis of the distribution of HOXB7 protein in 3T3 cells, and of HOXD4 protein in human neuroblastoma and Raji lymphoma cell lines and activated lymphocytes, results invariably in a nucleolar localisation. Purified nucleoli from stimulated T lymphocytes, and Raji cells contain an activity capable of binding, in a gel retardation assay, to an oligonucleotide specifically recognised by the HOXD4 homeoprotein. This activity is specifically removed by anti-HOXD4 antibodies and is found associated in southwestern blots with a single band with an apparent Mr of 30,000, corresponding to that of recombinant HOXD4. The functional significance of the nucleolar localisation of Hox proteins remains to be determined.
Activation of the small GTP-binding proteins rho and rac by growth factor receptors
Authors: Nobes Catherine D. , Hawkins Phillip , Stephens Len , Hall Alan
Published: April 2021

The small GTP-binding proteins, rho and rac, control signal transduction pathways that link growth factor receptors to the activation of actin polymerization. In Swiss 3T3 cells, rho proteins mediate the lysophosphatidic acid and bombesin-induced formation of focal adhesions and actin stress fibres, whilst rac proteins are required for the platelet-derived growth factor-, insulin-, bombesin- and phorbol ester (phorbol 12-myristate 13-acetate)-stimulated actin polymerization at the plasma membrane that results in membrane ruffling. To investigate the role of p85/p110 phosphatidylinositol 3-kinase in the rho and rac signalling pathways, we have used a potent inhibitor of this activity, wortmannin. Wortmannin has no effect on focal adhesion or actin stress fibre formation induced by lysophosphatidic acid, bombesin or microinjected recombinant rho protein.

In contrast, it totally inhibits plasma membrane edge-ruffling induced by platelet-derived growth factor and insulin though not by bombesin, phorbol ester or microin-jected recombinant rac protein. We conclude that phos-phatidylinositol 3,4,5 trisphosphate mediates activation of rac by the platelet-derived growth factor and insulin receptors. The effects of lysophosphatidic acid on the Swiss 3T3 actin cytoskeleton can be blocked by the tyrosine kinase inhibitor, tyrphostin. Since tyrphostin does not inhibit the effects of microinjected rho protein, we conclude that lysophosphatidic acid activation of rho is mediated by a tyrosine kinase.
A protein similar to the 67 kDa laminin binding protein and p40 is probably a component of the translational machinery in Urechis caupo oocytes and embryos
Authors: Rosenthal Eric T. , Wordeman Linda
Published: April 2021

Oocytes of the echiuroid, Urechis caupo, contain an abundant maternal mRNA that encodes a protein very similar to LBP/p40, originally identified as a non-integrin 67 kDa laminin binding protein. We have sequenced the Urechis caupo mRNA for LBP/p40, and a similar mRNA from the Hawaiian sea urchin, Tripneustes gratilla. Both of the encoded proteins, as well as LBP/p40 proteins from other sources, share significant homology in the amino 2/3 of the proteins, but diverge extensively at the carboxyl ends. LBP/p40 protein is present in growing and in full-grown U. caupo oocytes. The protein concentration remains constant for the first 48 hours of embryogenesis and then begins to decline. In sucrose gradients run with homogenates from coelomocytes, oocytes, and early embryos, all of the LBP/p40 protein appears to be associated with either polysomes or free 40 S ribosomal subunits. In later embryos, an increasing proportion of the protein is found in the soluble fraction. Immunohistochemistry indicates that LBP/p40 is uniformly distributed in early U. caupo embryos, with no localization at the cell surface. In later embryos LBP/p40 is localized in specific parts of the embryo which may correspond to neural tissue.
Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus
Authors: Dargemont Catherine , Schmidt-Zachmann Marion S. , Kühn Lukas C.
Published: April 2021

Primary transcripts in eukaryotic cells undergo several processing steps within the nucleus, and resulting mature RNA molecules are selectively exported to the cytoplasm. Nucleo-cytoplasmic mRNA transport is an active process that likely involves RNA-protein interactions. To identify specific RNA-binding proteins, we designed a novel approach, which allows the analysis of interactions between mRNAs and proteins along the transport pathway. The method consists of inducing in vivo a covalent binding between nuclear proteins and microinjected mRNAs. Using such a procedure, we were able to detect a direct interaction between nucleoporin p62 with mRNA during export. The formation of the mRNA-p62 complex was inhibited by wheat-germ agglutinin, an inhibitor of mRNA export. Antibodies directed against p62 caused a substantial reduction in the rate of mRNA export from the nucleus.
Identification of subcellular compartments containing peptidylglycine α-amidating monooxygenase in rat anterior pituitary
Authors: Oyarce Ana Maria , Eipper Betty A.
Published: April 2021

Both soluble and integral membrane forms of peptidylglycine α-amidating monooxygenase (PAM) are expressed in the rat anterior pituitary, making it an ideal model system for studying the routing of proteins into secretory granules. To identify the subcellular compartments involved in the routing of integral membrane PAM, we used subcellular fractionation, metabolic labeling and immunoblot analysis. Mature secretory granules were found to contain full-length integral membrane PAM along with a significant amount of soluble PAM generated by endoproteolytic cleavage. PAM proteins were not co-dis-tributed with tyrosylprotein sulfotransferase activity during sucrose gradient centrifugation, indicating that the trans-Golgi/TGN is not a major PAM-containing compart-ment at steady state. Fractionation of the 4,000 g and 10,000 g pellets obtained by differential centrifugation identified a significant amount of integral membrane PAM in a light fraction lacking soluble secretory granule proteins. Metabolic labeling experiments with primary anterior pituitary cells demonstrated that integral membrane PAM enters a light compartment with similar properties only after exit from the trans-Golgi/TGN. Com-parison of the metabolic labeling and immunoblot analyses suggests that PAM in this post-trans-Golgi/TGN compart-ment is in organelles involved in the intracellular recycling of integral membrane PAM. Small amounts of full-length integral membrane PAM were also recovered in fractions containing internalized transferrin and may be in an endosomal compartment following retrieval from the cell surface.
A dialysis culture system for the study of the production and modulation of growth-regulatory molecules: studies using the P388D1 macrophage cell line
Authors: Proudfoot D. , Parrott D. P. , Bowyer D. E.
Published: April 2021

P388D1 macrophage-like cells have previously been shown to produce both mitogenic and inhibitory regulators of porcine smooth muscle cell (pSMC) growth. The mitogenic activity was shown to have a molecular mass of >10 kDa while the inhibitory activity was in the range of 2-6 kDa. In the present study, we present a novel dialysis culture system where P388D1 cells were grown in dialysis membranes with a 12 kDa cut-off which allowed continu-ous production of fractions of the culture medium. Using pSMC as target cells, mitogenic activity was found to be retained by the dialysis membrane while the low molecular mass inhibitory activity passed freely through the membrane.

The effect of the macrophage-activators phorbol myristate acetate (PMA), concanavalin A (ConA) and interferon-γ in combination with lipopolysaccharide (IFNγ/LPS) were investigated in the dialysis culture system. PMA, ConA and IFNγ/LPS were found to enhance the production of mitogenic activity by P388D1 cells. PMA also increased the production of growth-inhibitory activity, while ConA abolished inhibitor production and IFNγ/LPS had no effect on the amount of inhibitory activity produced by P388D1 cells.

The experiments show that the balance of production of mitogenic and inhibitory activities by macrophages can be modulated by agents that alter the state of activation of the cells. This could be of profound significance in the influence of macrophages on smooth muscle cell growth during the development of atherosclerosis.
A mechanical function of myosin II in cell motility
Authors: Jay Patrick Y. , Pham Peter A. , Wong Scott A. , Elson Elliot L.
Published: April 2021

Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell’s rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild-type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a defect in retraction, pseudopod extension does not cause the cells to become elongated on sticky surfaces. This suggests a mechanism, based possibly on cytoskeletal tension, for regulating cell shape in locomotion. The tension would result from the transmission of tractional forces through the cytoskeletal network, providing the myosin II null mutant with a limited means of retraction and cell division on a surface.
In vivo expression of alternatively spliced forms of integrin-associated protein (cd47)
Authors: Reinhold Martina I. , Lindberg Frederik P. , Plas David , Reynolds Stacy , Peters Marion G. , Brown Eric J.
Published: April 2021

Integrin-associated protein (IAP) is a 50 kDa plasma membrane protein physically and functionally associated with β3 integrins in a variety of cells. IAP has an extracellular immunoglobulin domain, five transmembrane domains and a short intracytoplasmic tail. IAP is recognized by anti-CD47 antibodies and is expressed on cells, such as erythrocytes and lymphocytes, which do not express β3 integrins. To learn more about potential functions of IAP we examined its expression in vivo. Using the polymerase chain reaction, we detected 4 alternatively spliced forms of IAP which differ from each other only at their intracytoplasmic carboxy termini. These alternatively spliced forms are generated by inclusion or exclusion of three short exons within 5 kb in the genome and are highly conserved between mouse and man. There is tissue specificity of expression of the alternatively spliced forms of IAP mRNA, with bone marrow-derived cells expressing pre-dominantly one form and neural tissue another. Using polyclonal antibodies which recognize the alternatively spliced bone marrow (form 2) and neural (form 4) forms of IAP, we found that in accord with the mRNA, form 2 protein was expressed in all tissues primarily on bone marrow-derived cells and endothelia, while form 4 was highly expressed in the brain and peripheral nervous system. The evolutionary conservation of IAP isoforms and their tissue-specific expression suggest an important role for these intracytoplasmic domains in IAP function.
Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from ebs patients
Authors: Morley S. M. , Dundas S. R. , James J. L. , Gupta T. , Brown R. A. , Sexton C. J. , Navsaria H. A. , Leigh I. M. , Lane E. B.
Published: April 2021

Point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorders such as epidermolysis bullosa simplex, in which epidermal keratinocytes are extremely fragile and the skin blisters on mild trauma. We show that in 2 phenotypically diverse cases of epidermolysis bullosa simplex, the keratin mutations result in a thermoinstability of the intermediate filament cytoskeleton which can be reproducibly demonstrated even in the presence of tissue culture-induced keratins and in conditions where filament fragility is not otherwise obvious.

SV40-T antigen and HPV16 (E6/E7) immortalised keratinocyte cell lines were examined, established from control and epidermolysis bullosa simplex-affected individuals with either severe (Dowling-Meara) or mild (Weber-Cockayne) forms of the disease. In standard tissue culture conditions no significant and consistent abnormality of the keratin cytoskeleton could be demonstrated. However after thermal stress a reduced stability of the keratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines, with filaments breaking into aggregates similar to those seen in skin from EBS patients. These aggregates were maximal at 15 minutes after heat shock and the filament network structure was substantially reversed by 60 minutes.

Differences were also seen in the cells during respreading after replating: cells containing mutant keratins were slower to respread than controls and fine aggregates were seen at the cell margins in the Dowling-Meara derived cell line. Such delays in restoring the normal intermediate filament network after physiological processes involving cytoskeleton remodelling may render the cells vulnerable to cytolysis in vivo if physically challenged during this time window. The steady reduction in the mitotic index of the epidermis during the first few years of life could then explain the clinical improvement which is frequently observed in growing children.
Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: Implications for the role of dynamic microtubules in cell locomotion
Authors: Liao Guojuan , Nagasaki Takayuki , Gundersen Gregg G.
Published: April 2021

The role of microtubules (MTs) in cell locomotion is uncertain: while MTs are not essential for motility of certain cells, MTs are necessary for the directed translocation of large cells such as fibroblasts, endothelial cells and neuronal growth cones. Based on previous studies, we hypothesize that cell locomotion may involve MTs in two possible ways: (1) the rate of cell locomotion is proportional to MT level; or (2) cell locomotion is not proportional to MT level but requires a critical level of MTs to proceed. To test these hypotheses, we measured the rate of locomotion of NRK fibroblasts migrating into an in vitro wound, before and after treatment with different concentrations of nocodazole to generate cells with different levels of MTs. Locomotion of cells was monitored directly using timelapse recording and analyzed with an Image-1 image analysis program. Addition of nocodazole (≥50 nM) resulted in a rapid reduction in locomotion to a new rate that was maintained for >2 hours. We found that addition of as little as 100 nM nocodazole decreased the rate of locomotion by more than 60%; and that 300 nM nocodazole completely stopped cell locomotion. Although 100 nM nocodazole decreased locomotion over 60%, we detected no qualitative change in MT distribution by immunofluorescence. Quantitative analysis of MT fluorescence in immunofluorescently stained preparations showed that 100 nM nocodazole had no detectable effect on MT levels and that 300 nM nocodazole only decreased MT levels to ∼40% of controls. Quantitative analysis of tubulin polymer levels by cell extraction and western blotting yielded results similar to those obtained by quantification of MT fluorescence. A comparison of the locomotion rate measurements with the MT level measurements indicated that over half of the cell locomotion rate could be blocked by nocodazole without significantly affecting MT levels in the cell; the remaining locomotion rate was reduced proportionally to MT levels. These results do not support the notion that a critical level of MTs is required for cell locomotion and suggest that only a portion (<50%) of the speed of the cells is proportional to MT levels. Rather, by analogy with studies of MT antagonists on the mitotic spindle, they suggest a third possibility: that low concentrations of nocodazole interfere with MT dynamics and thus, MT dynamics are critical for the maximal speed of cell locomotion. This notion was further supported by analogous effects of taxol and vinblastine on cell locomotion: at concentrations that reportedly cause little change in the level of MTs, taxol and vinblastine also dramatically decreased the rate of locomotion of NRK cells. In summary, our results establish the relationship between microtubule levels and locomotion rate and suggest that dynamic MTs are rate-limiting for fibroblast locomotion.
Clustering of cx43 cell-to-cell channels into gap junction plaques: Regulation by camp and microfilaments
Authors: Wang Yingjian , Rose Birgit
Published: April 2021

Cell-to-cell channels are often seen clustered at cell-cell contacts into the so-called gap junction plaques. The mechanism of this clustering is unknown. We show that the clustering of cell-to-cell channels composed of connexin43 is induced by elevation of cyclic AMP. The cAMP-induced clustering is enhanced by inhibition of glycosylation and abolished by disruption of microfilaments. Channel clustering thus seems to be regulated by cAMP and glycosylation and to involve microfilaments.
Characterisation of neurofilament protein nf70 mrna from the gastropod Helix aspersa reveals that neuronal and non-neuronal intermediate filament proteins of cerebral ganglia arise from separate lamin-related genes
Authors: Adjaye James , Plessmann Uwe , Weber Klaus , Dodemont Huub
Published: April 2021

The neuronal cytoplasmic intermediate filament (IF) protein HeNF70 of the gastropod Helix aspersa is identified by sequence analysis of the corresponding 4,600 bp cDNA isolated from a cerebral ganglion cDNA library. HeNF70 shares 60% sequence identity with the neuronal LoNF70 protein of the cephalopod Loligo pealei and only 36% identity with the Helix non-neuronal IF-A protein. All three molluscan IF proteins display the lamin-type extended coil 1b subdomain harbouring six additional heptads and all have long C-terminal sequences with sub-stantial homology to the tail domains of nuclear lamins. The lamin-like tail domains of the two neurofilament proteins share a unique motif comprising 13 residues, which is absent from Helix IF-A and all other known non-neuronal IF proteins. HeNF70 is encoded by a 9.5 kb RNA transcript. The very long 7.2 kb 3′-untranslated sequence contains a unique 26 nucleotide repeat extending over 0.5 kb in its 5′-region. The HeNF70 mRNA is expressed at low abundancy in cerebral ganglia but not in any of the 13 non-neuronal tissues tested. In contrast, all tissues express at fairly high levels the same 4.6 and 4.2 kb mRNAs encoding the Helix non-neuronal IF-A/B proteins. Blot hybridisation studies on genomic DNA and ganglion mRNA with subprobes from the cloned HeNF70 cDNA, as well as sequence analysis of an RT-PCR generated partial cDNA encoding a putative HeNF60 protein, indicate at least two different neuronal IF genes in Helix.
Characterisation of a laminarin sulphate which inhibits basic fibroblast growth factor binding and endothelial cell proliferation
Authors: Hoffman Richard , Paper Dietrich H. , Donaldson Jane , Alban Susanne , Franz Gerhard
Published: April 2021

We have evaluated a series of laminarin sulphates with different degrees of sulphation (0.3-2.3) as antagonists of basic fibroblast growth factor (bFGF) and as inhibitors of the bFGF-dependent endothelial cell line FBHE. Inhibition of binding of bFGF by the laminarin sulphates increased with increasing degree of sulphation. Binding of bFGF to low affinity sites on BHK cells was inhibited more strongly than binding to high affinity sites. IC50 values for inhibi-tion of binding to low and high affinity sites by the most highly sulphated laminarin sulphate (LAM S5; degree of sulphation 2.31) were 12±8 µ g/ml and 69±66 µ g/ml, respec-tively. LAM S5 dissociated bFGF from low affinity sites on BHK cells but not from high affinity sites. LAM S5 increased the electrophoretic mobility of bFGF indicating that LAM S5 binds directly to bFGF. LAM S5 reduced uptake of bFGF by FBHE cells by 67%. Increasing the degree of sulphation of laminarin sulphates increased the inhibition of bFGF-stimulated DNA synthesis of the endo-thelial cell line FBHE (IC50 for LAM S5 approx. 1 µ g/ml). There was no inhibition of DNA synthesis of FBHE cells by LAM S5 in the presence of 1 µ g/ml bFGF indicating that bFGF antagonism is involved in the anti-proliferative activity of this compound. LAM S5 may be of value against diseases associated with bFGF-dependent cell proliferation.
Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in african trypanosomes
Authors: Brickman Marla Jo , Cook J. Michael , Balber Andrew E.
Published: April 2021

We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of fluid phase bovine serum albumen gold complexes and membrane bound concanavalin A through endosomal com-partments of bloodstream forms of Trypanosoma brucei rhodesiense. Both markers were rapidly endocytosed from the flagellar pocket. Within 20 minutes at 37°C the markers reached a large, vesicular, perinuclear compartment that stained heavily with the CB1 monoclonal antibody. Neither marker left the flagellar pocket and entered cells at 4°C. When cells were incubated at 12°C, both markers entered the cell and were transported to collecting tubules, a tubular endosomal compartment that receives endocytosed material from coated endocytic vesicles. However, no material was transported from collecting tubules to the late, perinuclear compartment at 12°C. The morphology of collecting tubule membranes was specifically altered at 12°C; tubules became shorter and were arrayed near the flagellar pocket. The morphological alteration and the block in transport of endocytic markers to the perinuclear compartment seen at 12°C were reversed 10 minutes after cells were returned to 37°C. We also used flow cytofluori-metric measurements of pH dependent fluorescence quenching to measure the pH of the terminal endocytic compartment. Fluoresceinated lectins accumulated in a terminal compartment with a pH of 6.0-6.1, a value con-siderably higher than that of mammalian lysosomes. Flu-orescence from fluoresceinated lectins in this terminal endocytic compartment was dequenched when blood-stream forms were incubated in the presence of chloro-quine.