bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic control of peroxisome abundance
Authors: Chang Chia-Che , South Sarah , Warren Dan , Jones Jacob , Moser Ann B. , Moser Hugo W. , Gould Stephen J.
Published: April 2021

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal β-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal β-oxidation activities suggests that there may be metabolic control of peroxisome abundance.
Alternative mechanisms for trafficking of lysosomal enzymes in mannose 6-phosphate receptor-deficient mice are cell type-specific
Authors: Dittmer Frank , Ulbrich Eva J. , Hafner Angela , Schmahl Wolfgang , Meister Tobias , Pohlmann Regina , Figura Kurt von
Published: April 2021

Viable mice nullizygous in genes encoding the 300 kDa and the 46 kDa mannose 6-phosphate receptors (MPR 300 and MPR 46) and the insulin like growth factor II (IGF II) were generated to study the trafficking of lysosomal enzymes in the absence of MPRs. The mice have an I-cell disease-like phenotype, with increase of lysosomal enzymes in serum and normal activities in tissues. Surprisingly, the ability of MPR-deficient cells to transport newly synthesized lysosomal enzymes to lysosomes and the underlying mechanisms were found to depend on the cell type. MPR-deficient thymocytes target newly synthesized cathepsin D to lysosomes via an intracellular route. In contrast, hepatocytes and fibroblasts secrete newly synthesized cathepsin D. In fibroblasts recapture of secreted lysosomal enzymes, including that of cathepsin D, is limited and results in lysosomal storage, both in vivo and in vitro, whereas recapture by hepatocytes is remarkably effective in vivo and can result in lysosomal enzyme levels even above normal.
The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro
Authors: Kräling Birgit M. , Wiederschain Dmitri G. , Boehm Thomas , Rehn Marko , Mulliken John B. , Moses Marsha A.
Published: April 2021

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor ε-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.
Mechanism of extracellular domain-deleted dominant negative cadherins
Authors: Nieman Marvin T. , Kim Jae-Beom , Johnson Keith R. , Wheelock Margaret J.
Published: April 2021

The cadherin/catenin complex mediates Ca2+-dependent cell-cell interactions that are essential for normal developmental processes. It has been proposed that sorting of cells during embryonic development is due, at least in part, to expression of different cadherin family members or to expression of differing levels of a single family member. Expression of dominant-negative cadherins has been used experimentally to decrease cell-cell interactions in whole organisms and in cultured cells. In this study, we elucidated the mechanism of action of extracellular domain-deleted dominant-negative cadherin, showing that it is not cadherin isotype-specific, and that it must be membrane-associated but the orientation within the membrane does not matter. In addition, membranetargeted cytoplasmic domain cadherin with the cateninbinding domain deleted does not function as a dominantnegative cadherin. Expression of extracellular domain-deleted dominant-negative cadherin results in down-regulation of endogenous cadherins which presumably contributes to the non-adhesive phenotype.
The Golgi-targeting sequence of the peripheral membrane protein p230
Authors: Kjer-Nielsen Lars , Vliet Catherine van , Erlich Rebecca , Toh Ban-Hock , Gleeson Paul A.
Published: April 2021

Vesicle transport requires the recruitment of cytosolic proteins to specific membrane compartments. We have previously characterised a brefeldin A-sensitive trans-Golgi network-localised protein (p230) that is associated with a population of non-clathrin-coated vesicles. p230 recycles between the cytosol and the cytoplasmic face of buds/vesicles of trans-Golgi network membranes in a G protein-regulated manner. Identifying the mechanism responsible for Golgi targeting of p230 is important for the elucidation of its function. By transfection of COS cells with deletion mutants of p230 we here demonstrate that the C-terminal domain is necessary for targeting to the Golgi. Furthermore, the C-terminal 98 amino acid domain of p230 attached to the green fluorescent protein (GFP-p230-C98aa) was efficiently Golgi-localised in transfected COS cells. Deletion mutants of GFP-p230-C98aa together with alanine scanning mutagenesis identified a minimum stretch of 42 amino acids that is essential for Golgi targeting, suggesting that the conformation of the domain is critical for efficient targeting. In COS cells expressing high levels of GFP-p230-C98aa fusion protein, endogenous p230 was no longer associated with Golgi membranes, suggesting that the GFP fusion protein and endogenous p230 may compete for the same membrane target structures. The Golgi binding of GFP-p230-C98aa is brefeldin A-sensitive and is regulated by G proteins. These studies have identified a minimal sequence responsible for specific targeting of p230 to the Golgi apparatus, which displays similar membrane binding characteristics to wild-type p230.
Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane
Authors: Francis Michael J. , Jones Emma E. , Levy Elaine R. , Martin Rowena L. , Ponnambalam Sreenivasan , Monaco Anthony P.
Published: April 2021

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.
Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland
Authors: Metcalfe Anthony D. , Gilmore Andrew , Klinowska Teresa , Oliver Janine , Valentijn Anthony J. , Brown Robin , Ross Andrea , MacGregor Grant , Hickman John A. , Streuli Charles H.
Published: April 2021

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development.

To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways.

These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline.

Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.
Apical secretion of chondroitin sulphate in polarized Madin-Darby canine kidney (MDCK) cells
Authors: Kolset Svein Olav , Vuong Tram Thu , Prydz Kristian
Published: April 2021

Sugar moieties have been shown to contain sufficient and necessary information to target examples of secreted and transmembrane glycoproteins to the apical surface of epithelial MDCK cells. We have investigated if the sugar chains of proteoglycans, the glycosaminoglycans, also contain structural determinants for apical transport. Here we show that although 75% of the proteoglycan secretion from MDCK cell monolayers is into the basolateral medium, 75% of the proteoglycans of the chondroitin sulphate type are secreted apically. The sorting information in the chondroitin sulphate proteoglycans is localized to the sugar chains, since protein-free chondroitin sulphate chains, initiated on hexyl β-D-thioxyloside, were also predominantly secreted to the apical medium.
The polarization of the motile cell
Authors: Nabi Ivan R.
Published: April 2021

Polarization of the motile cell is associated with the formation of a distinct plasma membrane domain, the pseudopod, whose stabilization determines the directionality of cell movement. The rapid movement of cells over a substrate requires that an essential aspect of cell motility must be the supply of the necessary molecular machinery to the site of pseudopodial extension. Renewal of this pseudopodial domain requires the directed delivery to the site of pseudopodial protrusion of proteins which regulate actin cytoskeleton dynamics, cell-substrate adhesion, and localized degradation of the extracellular matrix. Polarized targeting mechanisms include the targeted delivery of β-actin mRNA to the leading edge and microtubule-based vesicular traffic. The latter may include Golgi-derived vesicles of the biosynthetic pathway as well as clathrin-dependent and clathrin-independent endocytosis and recycling. Coordination of protrusive activities and supply mechanisms is critical for efficient cellular displacement and may implicate small GTPases of the Rho family. While the specific molecular mechanisms underlying pseudopodial protrusion of the motile cell are well-characterized, discussion of these diverse mechanisms in the context of cellular polarization has been limited.
Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth
Authors: Seipel Katja , Medley Quintus G. , Kedersha Nancy L. , Zhang Xin A. , O’Brien Stephen P. , Serra-Pages Carles , Hemler Martin E. , Streuli Michel
Published: April 2021

Rho family GTPases regulate diverse cellular processes, including extracellular signal-mediated actin cytoskeleton reorganization and cell growth. The functions of GTPases are positively regulated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP. Trio is a complex protein possessing two guanine nucleotide exchange factor domains, each with adjacent pleckstrin homology and SH3 domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains. To assess the functional role of the two Trio guanine nucleotide exchange factor domains, NIH 3T3 cell lines stably expressing the individual guanine nucleotide exchange factor domains were established and characterized. Expression of the amino-terminal guanine nucleotide exchange factor domain results in prominent membrane ruffling, whereas cells expressing the carboxy-terminal guanine nucleotide exchange factor domain have lamellae that terminate in miniruffles. Moreover, cells expressing the amino-terminal guanine nucleotide exchange factor domain display more rapid cell spreading, haptotactic cell migration and anchorage-independent growth, suggesting that Trio regulates both cell motility and cell growth. Expression of full-length Trio in COS cells also alters actin cytoskeleton organization, as well as the distribution of focal contact sites. These findings support a role for Trio as a multifunctional protein that integrates and amplifies signals involved in coordinating actin remodeling, which is necessary for cell migration and growth.
Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis
Authors: Naumov George N. , Wilson Sylvia M. , MacDonald Ian C. , Schmidt Eric E. , Morris Vincent L. , Groom Alan C. , Hoffman Robert M. , Chambers Ann F.
Published: April 2021

High resolution intravital videomicroscopy has provided a powerful tool for directly observing steps in the metastatic process, and for clarifying molecular mechanisms of metastasis and modes of action of anti-metastasis therapeutics. Cells previously have been identified in vivo using exogenously added fluorescent labels, limiting observations to a few cell divisions, or by natural markers (e.g. melanin) expressed only by specific cell types. Here we tested the utility of stable green fluorescent protein (GFP)-transfected cells for monitoring and quantifying sequential steps in the metastatic process. Using CHO-K1 cells that stably express GFP, we document the visualization and quantification by intravital videomicroscopy of sequential steps in metastasis within mouse liver, from initial arrest of cells in the microvasculature to the growth and angiogenesis of metastases. Individual, non-dividing cells, as well as micro- and macrometastases could clearly be detected and quantified, as could fine cellular details such as pseudopodial projections, even after extended periods of in vivo growth. We quantified the size distribution of micrometastases and their locations relative to the liver surface using 50 μm thick formalin-fixed tissue sections. The data suggest preferential growth and survival of micrometastases near the liver surface. Furthermore, we observed a small population of single cells that persisted over the 11 day observation period, which may represent dormant cells with potential for subsequent proliferation. This study demonstrates the advantages of GFP-expressing cells, coupled with real-time high resolution videomicroscopy, for long-term in vivo studies to visualize and quantify sequential steps of the metastatic process.
Transcription, biochemistry and localization of nematode annexins
Authors: Daigle Scott N. , Creutz Carl E.
Published: April 2021

The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs. The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization. Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate. Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 μM calcium. Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 μM calcium. This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium. Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection. Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions. A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein. Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve.
Vascular-endothelial-cadherin modulates endothelial monolayer permeability
Authors: Hordijk Peter L. , Anthony Eloise , Mul Frederik P. J. , Rientsma Ronald , Oomen Lauran C. J. M. , Roos Dirk
Published: April 2021

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration.

In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the β2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than ‘jagged’, VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.
Human sweat gland myoepithelial cells express a unique set of cytokeratins and reveal the potential for alternative epithelial and mesenchymal differentiation states in culture
Authors: Schön Margarete , Benwood Jennifer , O’Connell-Willstaedt Therese , Rheinwald James G.
Published: April 2021

We have characterized precisely the cytokeratin expression pattern of sweat gland myoepithelial cells and have identified conditions for propagating this cell type and modulating its differentiation in culture. Rare, unstratified epithelioid colonies were identified in cultures initiated from several specimens of full-thickness human skin. These cells divided rapidly in medium containing serum, epidermal growth factor (EGF), and hydrocortisone, and maintained a closely packed, epithelioid morphology when co-cultured with 3T3 feeder cells. Immunocytochemical and immunoblot analysis disclosed that the cells differed from keratinocytes in that they were E-cadherin-negative, vimentin-positive, and expressed an unusual set of cytokeratins, K5, K7, K14, and K17. When subcultured without feeder cells, they converted reversibly to a spindle morphology and ceased K5 and K14 expression. Under these conditions, EGF deprivation induced flattening, growth arrest, and expression of α-smooth muscle actin (α-sma). Coexpression of keratins and α-sma is a hallmark of myoepithelial cells, a constituent of secretory glands. Immunostaining of skin sections revealed that only sweat gland myoepithelial cells expressed the same pattern of keratins and α-sma and lack of E-cadherin as the cell type we had cultured. Interestingly, our immunocytochemical analysis of ndk, a skin-derived cell line of uncertain identity, suggests that this line is of myoepithelial origin. Earlier immunohistochemical studies by others had found myoepithelial cells to be K7-negative. We tested five K7-specific antibodies that can recognize this protein in western blots and in the assembled keratin filaments of mesothelial cells. Three of these antibodies did not recognize the K7 present in myoepithelial cell filaments or in HeLa cell filaments, indicating that some K7 epitopes are masked when K7 pairs with K17 instead of with its usual keratin filament partner, K19.
Sto1p, a fission yeast protein similar to tubulin folding Cofactor E, plays an essential role in mitotic microtubule assembly
Authors: Grishchuk Ekaterina L. , McIntosh J. Richard
Published: April 2021

The proper functioning of microtubules depends crucially on the availability of polymerizable α/β tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1+, a gene that encodes a protein similar to Cofactor E, which is required for the folding of α-tubulin. The interphase microtubule cytoskeleton in sto1-Δ cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the α-but not the β-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt α/β tubulin dimers in vivo.
Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration
Authors: Bird Ian N. , Taylor Vanessa , Newton Justin P. , Spragg Julia H. , Simmons David L. , Salmon Michael , Buckley Christopher D.
Published: April 2021

PECAM-1 (CD31) is a highly abundant cell surface glycoprotein expressed on haemopoietic and endothelial cells. As well as mediating homophilic (PECAM-1/PECAM-1) adhesion, PECAM-1 can also bind the integrin αvβ3. Both PECAM-1 and αvβ3 have been shown to have roles in regulating angiogenesis, endothelial tube formation and in the case of αvβ3, endothelial cell apoptosis. In this study we show that despite being expressed at equivalent levels, endothelial αvβ3 is not a ligand for PECAM-1. Rather, PECAM-1 supports homophilic binding on HUVEC with similar characteristics to those we have previously reported for leukocytes and becomes tyrosine phosphorylated after homophilic PECAM-1 and integrin/fibronectin engagement. Immunoprecipitation studies show that in addition to SHP-2, tyrosine phosphorylated PECAM-1 can interact with at least four other phosphoproteins in pervanadate stimulated HUVEC. While PECAM-1/PECAM-1 interactions support robust endothelial cell adhesion, they do not support cell spreading or migration. In addition PECAM-1 homophilic adhesion rescues HUVEC from serum deprivation-induced apoptosis. Taken together our results indicate that PECAM-1 homophilic interactions play an important role in interendothelial cell adhesion, survival and signalling.
A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA
Authors: Ludtke James J. , Zhang Guofeng , Sebestyén Magdolna G. , Wolff Jon A.
Published: April 2021

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310-1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.
Insect cuticle, an in vivo model of protein trafficking
Authors: Csikós György , Molnár Kinga , Borhegyi Noémi H. , Talián Gábor Cs. , Sass Miklós
Published: April 2021

In the course of this study more than 20 proteins have been isolated from the larval cuticle of Manduca sexta. Synthesis, secretion, transport and accumulation of four particular proteins, representative members of four characteristic groups, were followed during metamorphosis by immunoblot and immuncytochemical methods and are described in detail in this paper. We established that only some of the proteins of the soft cuticle of Lepidopteran larvae are synthesized in epidermal cells at the beginning of the larval stages and are digested during the moulting period (MsCP29). Other proteins (MsCP30/11) are secreted into the cuticle by the epidermal cells in different forms during various developmental stages. Some proteins are secreted apically during the feeding period, but before ecdysis they are then taken up by epidermal cells and transported in a basolateral direction back into the hemolymph and saved in an immunologically intact form by the fat body cells (MsCP12.3). Some cuticle proteins have a non-epidermal origin. They are transported from the hemolymph into the cuticle. Before and during ecdysis these molecules reappear in the hemolymph and are detectable again in the pupal cuticle (MsCP78). Our data prove that the cuticle is not a non-living part of the insect body: it is not only an inert, protective armor, but maintains a continuous and dynamic metabolic connection with the other organs of the organism.
3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription
Authors: Panse Sophie Le , Masson Claude , Héliot Laurent , Chassery Jean-Marc , Junéra Henriette Roberte , Hernandez-Verdun Danièle
Published: April 2021

In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e. the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace. This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functional units. Transcript labeling was not similar for each unit, contrary to UBF labeling. UBF and rRNA transcripts were only partially colocalized, as demonstrated by 3-D image analysis and quantification. As visualized by electron microscopy, the necklace was composed of a small fibrillar center partially surrounded by a dense fibrillar component. The 3-D arrangement of this individual unit in the necklace, investigated both by confocal and electron microscopy in the same cells, showed that the individual beads were linked by a dense fibrillar component. The reversibility of this organization after removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these results lead us to suggest that the organization of a functional domain, presumably corresponding to a single gene, can be studied by in situ approaches.
Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages
Authors: Wassenberg James J. , Dezfulian Cameron , Nicchitta Christopher V.
Published: April 2021

Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elicits cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell-derived peptides. Elicitation of a CTL response requires that GRP94-associated peptides be transferred onto major histocompatability complex (MHC) class I molecules, a process that is postulated to accompany GRP94 internalization by antigen presenting cells, such as macrophages (MΦ) and dendritic cells (DC). In studies of GRP94 uptake in elicited MΦ, we report that MΦ display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis. GRP94 internalized by this pathway co-localized predominately with transferrin-positive early endosomes. At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed. When GRP94 was present in the medium, and thus accessible to both receptor-mediated and fluid phase internalization pathways, internalization was modestly inhibited in the presence of yeast mannan, a competitive inhibitor of mannose/fucose receptor activity, and substantially inhibited by dimethylamiloride, an inhibitor of macropinocytosis. GRP94 internalized via macropinocytosis did not display prominent co-staining with the lysosomal marker LAMP-2. These data identify multiple pathways of GRP94 internalization and indicate that receptor-dependent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety. Most significantly, these data demonstrate the existence of cell surface receptor(s), apparently unique to antigen presenting cells, that function in the binding and internalization of the ER chaperone GRP94.
RHO-associated protein kinase α potentiates insulin-induced MAP kinase activation in Xenopus oocytes
Authors: Ohan Nicholas , Agazie Yehenew , Cummings Cathy , Booth Ronald , Bayaa Mustafa , Johné Liu X.
Published: April 2021

We recently identified Xenopus Rho-associated protein kinase α (xROKα) as a Xenopus insulin receptor substrate-1 binding protein and demonstrated that the non-catalytic carboxyl terminus of xROKα binds Xenopus insulin receptor substrate-1 and blocks insulin-induced MAP kinase activation and germinal vesicle breakdown in Xenopus oocytes. In the current study we further examined the role of xROKα in insulin signal transduction in Xenopus oocytes. We demonstrate that injection of mRNA encoding the xROKα kinase domain or full length xROKα enhanced insulin-induced MAP kinase activation and germinal vesicle breakdown. In contrast, injection of a kinase-dead mutant of xROKα or pre-incubation of oocytes with an xROKα inhibitor significantly reduced insulin-induced MAP kinase activation. To further dissect the mechanism by which xROKα may participate in insulin signalling, we explored a potential function of xROKα in regulating cellular Ras function, since insulin-induced MAP kinase activation and germinal vesicle breakdown is known to be a Ras-dependent process. We demonstrate that whereas injection of mRNA encoding c-H-Ras alone induced xMAP kinase activation and GVBD in a very low percentage (about 10%) of injected oocytes, co-injection of mRNA encoding xROKα and c-H-Ras induced xMAP kinase activation and germinal vesicle breakdown in a significantly higher percentage (50-60%) of injected oocytes. These results suggest a novel function for xROKα in insulin signal transduction upstream of cellular Ras function.
The Drosophila forked protein induces the formation of actin fiber bundles in vertebrate cells
Authors: Grieshaber Scott , Petersen Nancy S.
Published: April 2021

The forked protein is an actin binding protein involved in the formation of large actin fiber bundles in developing Drosophila bristles. These are the largest example of a type of actin bundle characterized by parallel, hexagonally packed actin fibers, also found in intestinal microvilli, kidney proximal tubule microvilli, and stereocilia in the ear. Understanding how these structures are constructed and how that construction is regulated is an important question in cell and developmental biology. Because the timing of forked gene expression coincides with the formation of the actin fiber bundles, and since the forked protein is localized at the site of initiation of these bundles before they form, it has been proposed that the forked protein is an initiator of actin bundle formation. In this paper we show that the forked protein can induce the formation of bundles and increase actin polymerization in vertebrate cells. We use this system to identify regions of the forked protein which are essential for bundle formation and actin co-localization.
Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D
Authors: Rothbarth Karsten , Spiess Eberhard , Juodka Benediktas , Yavuzer Ugur , Nehls Peter , Stammer Hermann , Werner Dieter
Published: April 2021

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect.

C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.
Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins
Authors: Ching Gee Y. , Liem Ronald K. H.
Published: April 2021

Type IV neuronal intermediate filament proteins consist of α-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat α-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lαα and Mαα, the chimeric proteins generated by replacing the head domain of α-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, αLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of α-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the α-internexin head domain is essential for α-internexin’s ability to self-assemble. While coassembly of Lαα with NF-M and coassembly of Mαα with NF-L resulted in formation of filaments, coassembly of Lαα with NF-L and coassembly of Mαα with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.
Contribution of plus and minus end pathways to microtubule turnover
Authors: Vorobjev I. A. , Rodionov V. I. , Maly I. V. , Borisy G. G.
Published: April 2021

Turnover is important for the maintenance and remodeling of the cytoskeleton during the processes of cell morphogenesis, mitosis and motility. Microtubule (MT) turnover is thought to occur by dynamic instability, growth and shortening at distal (plus) ends. Recent observation of MT release from the centrosome and depolymerization from proximal (minus) ends indicates the existence of a minus end pathway. To evaluate the relative contributions of plus and minus end pathways to turnover, we analyzed MT dynamics in a model system, the fish melanophore, a large non-motile cell with a regular radial array of long MTs. MT ends were tracked in digital fluorescence time-lapse sequences and life histories of individual MTs were analyzed using random walk theory generalized to the case of diffusion with drift. Analysis of plus end dynamics gave an apparent diffusion coefficient of D=7.5 μm2/minute. The random walk model predicts that the half-time for turnover driven solely by plus end dynamics will depend strongly on position in the cell. Based on the experimentally determined value of D, turnover of MTs near the center of a typical melanophore of radius 70 μm was calculated to require over 5 hours, a paradoxically long time.

To examine MT behavior deep in the cytoplasm, we developed a novel, sequential subtraction mode of image analysis. This analysis revealed a subpopulation of MTs which shortened from their minus ends, presumably after constitutive release from the centrosome. Given the relative slowness of plus end dynamics to turn over the root of a long MT, the turnover of MTs near the cell center is determined primarily by the minus-end pathway. MTs released from the centrosome become replaced by newly nucleated ones. The relative contributions of plus and minus end pathways was estimated from the diffusion coefficient, D, for the plus end, the length distribution of MTs, t he frequency of free minus ends, and the rate of minus-end shortening. We conclude that, in large animal cells with a centrosomally focussed array of MTs, turnover occurs by a combination of plus and minus end pathways, the plus end dominating at the cell periphery and the minus end dominating near the cell center.
Innexin-3 forms connexin-like intercellular channels
Authors: Landesman Yosef , White Thomas W. , Starich Todd A. , Shaw Jocelyn E. , Goodenough Daniel A. , Paul David L.
Published: April 2021

Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels.
p53-independent apoptosis induced by muscle differentiation stimuli in polyomavirus large T-expressing myoblasts
Authors: Gottifredi Vanesa , Peschiaroli Angelo , Fimia Gian Maria , Maione Rossella
Published: April 2021

Abnormal proliferation signals, driven by cellular or viral oncogenes, can result in the induction of apoptosis under sub-optimal cell growth conditions. The tumor suppressor p53 plays a central role in mediating oncogene-induced apoptosis, therefore transformed cells lacking p53 are generally resistant to apoptosis-promoting treatments. In a previous work we have reported that the expression of polyomavirus large T antigen causes apoptosis in differentiating myoblasts and that this phenomenon is dependent on the onset of muscle differentiation in the absence of a correct cell cycle arrest. Here we report that polyomavirus large T increases the levels and activity of p53, but these alterations are not involved in the apoptotic mechanism. Apoptosis in polyomavirus large T-expressing myoblasts is not prevented by the expression of a p53 dominant-negative mutant nor it is increased by p53 over-expression. Moreover, forced differentiation induced through the over-expression of the muscle regulatory factor MyoD, leads to apoptosis without altering p53 function and, more significantly, even in a p53-null background. Our results indicate that apoptosis induced by the activation of muscle differentiation pathways in oncogene-expressing cells can occur in a p53-independent manner.
Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts
Authors: Zhang Chuanmao , Hughes Mike , Clarke Paul R.
Published: April 2021

Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.