bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of clathrin-coated pit assembly by an Eps15 mutant
Authors: Benmerah Alexandre , Bayrou Marie , Cerf-Bensussan Nadine , Dautry-Varsat Alice
Published: April 2021

Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of α-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.
A switch in the cellular localization of macrophage migration inhibitory factor in the rat testis after ethane dimethane sulfonate treatment
Authors: Meinhardt Andreas , Bacher Michael , O’Bryan Moira K. , McFarlane James R. , Mallidis Con , Lehmann Christof , Metz Christine N. , Kretser David M. de , Bucala Richard , Hedger Mark P.
Published: April 2021

Macrophage migration inhibitory factor (MIF), one of the first cytokines to be discovered, has recently been localized to the Leydig cells in adult rat testes. In the following study, the response of MIF to Leydig cell ablation by the Leydig cell-specific toxin ethane dimethane sulfonate (EDS) was examined in adult male rats. Testicular MIF mRNA and protein in testicular interstitial fluid measured by ELISA and western blot were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. Immunohistochemistry using an affinity-purified anti-mouse MIF antibody localized MIF exclusively to the Leydig cells in control testes. At 7 days post-EDS treatment, there were no MIF immunopositive Leydig cells in the interstitium, although distinct MIF immunostaining was observed in the seminiferous tubules, principally in Sertoli cells and residual cytoplasm, and some spermatogonia. A few peritubular and perivascular cells were also labelled at this time, which possibly represented mesenchymal Leydig cell precursors. At 14 and 21 days, Sertoli cell MIF immunoreactivity was observed in only a few tubule cross-sections, while some peritubular and perivascular mesenchymal cells and the re-populating immature Leydig cells were intensely labeled. At 28 days after EDS-treatment, the MIF immunostaining pattern was identical to that of untreated and control testes. The switch in the compartmentalization of MIF protein at 7 days after EDS-treatment was confirmed by western blot analysis of interstitial tissue and seminiferous tubules separated by mechanical dissection. These data establish that Leydig cell-depleted testes continue to produce MIF, and suggest the existence of a mechanism of compensatory cytokine production involving the Sertoli cells. This represents the first demonstration of a hitherto unsuspected pattern of cellular interaction between the Leydig cells and the seminiferous tubules which is consistent with an essential role for MIF in male testicular function.
HP33: hepatocellular carcinoma-enriched 33-kDa protein with similarity to mitochondrial N-acyltransferase but localized in a microtubule-dependent manner at the centrosome
Authors: Nakadai Tomoyoshi , Kishimoto Toshihiko , Miyazawa Yaeko , Okada Nami , Makino Yasutaka , Obinata Takashi , Tamura Taka-aki
Published: April 2021

Using a new subtraction method and chemically induced rat hepatocellular carcinomas, we identified a hepatocellular carcinogenesis and hepatocyte proliferation-related gene designated hp33 that encoded a 33-kDa protein. The predicted protein was similar to the bovine aralkyl N-acyltransferase and arylacetyl N-acyltransferase. HP33 was restrictively expressed in the liver and kidney, and its gene expression was stimulated in the regenerating liver as well as in hepatocellular carcinoma. Interestingly, it was demonstrated in various hepatic cells that HP33 was localized in regions surrounding the centrosome, where mitochondria were not concentrated. Moreover, its centrosomal localization was evident in the interphase but not in the mitotic phase of the cell cycle. The centrosomal localization of HP33 was dependent on microtubules, and ectopically expressed HP33 was seen at centrosomes even in fibroblasts, which do not exhibit a typical staining pattern of HP33. The centrosomal localization of HP33 became invisible by nocodazole treatment, whereas the mitochondrial staining pattern was not affected by it. In vitro cosedimentation experiments using purified microtubules indicated that HP33 bound to MTs directly and that its MT-binding ability was dependent on the C-terminal basic domain of the protein. These results suggest that, different from early predictions based on its primary structure, HP33 has a growthand carcinogenesis-related function that may be independent of mitochondrial function.
Glutathione S-conjugate transport in hepatocytes entering the cell cycle is preserved by a switch in expression from the apical MRP2 to the basolateral MRP1 transporting protein
Authors: Roelofsen Han , Hooiveld Guido J. E. J. , Koning Hans , Havinga Rick , Jansen Peter L. M. , Müller Michael
Published: April 2021

The multidrug resistance protein MRP1 and its isoform MRP2 are involved in ATP-dependent glutathione S-conjugate transport and have similar substrate specificities. MRP2 mediates hepatic organic anion transport into bile. The physiological function of MRP1 in hepatocytes is unknown. Previous results show that MRP1 expression is low in quiescent hepatocytes but increased after SV40 large T antigen immortalization, suggesting a relationship with cell proliferation. Therefore, we determined mrp1 and mrp2 expression in rat hepatocytes in relation to the cell cycle. By varying cell density we obtained cultures that are mainly in G1 (high density) or have progressed into the S-phase or beyond (low density). In both cultures mrp1 mRNA and protein levels are increased, concomitantly with the disappearance of mrp2. This switch from mrp2 to mrp1 occurs in the G1 phase of the cell cycle and is associated with a decreased cell polarity. Mrp1 is located on lateral membranes or on intracellular vesicles, depending on whether cell-cell contact is established. In both locations mrp1 contributes to cellular glutathione S-conjugate efflux and protects against oxidative stress-inducing quinones. We conclude that a switch in expression from the apically located mrp2 to the basolaterally located mrp1 preserves glutathione S-conjugate transport in hepatocytes entering the cell cycle and protects against certain cytotoxic agents.
Plastid tubules of higher plants are tissue-specific and developmentally regulated
Authors: Köhler Rainer H. , Hanson Maureen R.
Published: April 2021

Green fluorescent stroma filled tubules (stromules) emanating from the plastid surface were observed in transgenic plants containing plastid-localized green fluorescent protein (GFP). These transgenic tobacco plants were further investigated by epifluorescence and confocal laser scanning microscopy (CSLM) to identify developmental and/or cell type specific differences in the abundance and appearance of stromules and of plastids. Stromules are rarely seen on chlorophyll-containing plastids in cell types such as trichomes, guard cells or mesophyll cells of leaves. In contrast, they are abundant in tissues that contain chlorophyll-free plastids, such as petal and root. The morphology of plastids in roots and petals is highly dynamic, and plastids are often elongated and irregular. The shapes, size, and position of plastids vary in particular developmental zones of the root. Furthermore, suspension cells of tobacco exhibit stromules on virtually every plastid with two major forms of appearance. The majority of cells show a novel striking ‘octopusor millipede-like’ structure with plastid bodies clustered around the nucleus and with long thin stromules of up to at least 40 μm length stretching into distant areas of the cell. The remaining cells have plastid bodies distributed throughout the cell with short stromules. Photobleaching experiments indicated that GFP can flow through stromules and that the technique can be used to distinguish interconnected plastids from independent plastids.
Protein phosphatases PP1 and PP2A are located in distinct positions in the Chlamydomonas flagellar axoneme
Authors: Yang Pinfen , Fox Laura , Colbran Roger J. , Sale Winfield S.
Published: April 2021

We postulated that microcystin-sensitive protein phosphatases are integral components of the Chlamydomonas flagellar axoneme, positioned to regulate inner arm dynein activity. To test this, we took a direct biochemical approach. Microcystin-Sepharose affinity purification revealed a prominent 35-kDa axonemal protein, predicted to be the catalytic subunit of type-1 protein phosphatase (PP1c). We cloned the Chlamydomonas PP1c and produced specific polyclonal peptide antibodies. Based on western blot analysis, the 35-kDa PP1c is anchored in the axoneme. Moreover, analysis of flagella and axonemes from mutant strains revealed that PP1c is primarily, but not exclusively, anchored in the central pair apparatus, associated with the C1 microtubule. Thus, PP1 is part of the central pair mechanism thatcontrols flagellar motility. Two additional axonemal proteins of 62 and 37 kDa were also isolated using microcystin-Sepharose affinity. Based on direct peptide sequence and western blots, these proteins are the A-and C-subunits of type 2A protein phosphatase (PP2A). The axonemal PP2A is not one of the previously identified components of the central pair apparatus, outer arm dynein, inner arm dynein, dynein regulatory complex or the radial spokes. We postulate PP2A is anchored on the doublet microtubules, possibly in position to directly control inner arm dynein activity.
Intracellular targeting and retention of the glucose transporter GLUT4 by the perinuclear storage compartment involves distinct carboxyl-tail motifs
Authors: Martinez-Arca Sonia , Lalioti Vassiliki S. , Sandoval Ignacio V.
Published: April 2021

The mechanisms by which the insulin-sensitive glucose transporter, GLUT4, is targeted and retained in a storage compartment near to the Golgi complex are poorly understood. Here we report that removal of the carboxyl-terminal acidic Pro505AspGluAsnAsp509 sequence prevents the storage of GLUT4 in the VAMP-2 positive compartment adjacent to the Golgi complex (GSC), and results in its targeting to GLUT4-positive vesicles and Rab7-positive late endosomes. Storage of the truncated GLUT4 in the GSC is restored by substitution of Phe for the Tyr502 residue adjacent to Pro505 or by treatment of cells with the tyrosine kinase inhibitor genistein. Ablation of the Leu489Leu490-based motif prevents the targeting of GLUT4Δ5 to GLUT4-positive-vesicles and late endosomes as well as the retention of GLUT4Δ5Phe502 by the GSC.

These results are consisting with a model of GLUT4 transport in which the targeting of the protein from the TGN to the GSC is mediated by the Leu489Leu490-based motif and its release from the GSC involves Tyr502 and the adjacent carboxyl-terminal Pro505AspGluAsnAsp509 sequence.
Adhesion-dependent tyrosine phosphorylation of β-dystroglycan regulates its interaction with utrophin
Authors: James M. , Nuttall A. , Ilsley J. L. , Ottersbach K. , Tinsley J. M. , Sudol M. , Winder S. J.
Published: April 2021

Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated

β-dystroglycan and utrophin from cell extracts. Western blotting of β-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and β-dystroglycan by co-immunoprecipitation with endogenous β-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous β-dystroglycan was tyrosine phosphorylated, β-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide ‘SPOTs’ assays confirmed that tyrosine phosphorylation of β-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y892 in the β-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the β-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.
Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication
Authors: Gräf Ralph , Daunderer Christine , Schliwa Manfred
Published: April 2021

A cDNA encoding a 224-kDa Dictyostelium discoideumcentrosomal protein (DdCP224) was isolated by immunoscreening. DdCP224 was detected at the centrosome and, more weakly, along microtubules throughout the entire cell cycle. Centrosomal localization does not require microtubules, suggesting that DdCP224 is a genuine centrosomal component. DdCP224 exhibits sequence identity to a weakly conserved class of microtubule-associated proteins including human TOGp and yeast Stu2p. Stu2p has a size of only ∼100 kDa and corresponds to the N-terminal half of DdCP224. The functions of the N- and C-terminal halves of DdCP224 were investigated in the corresponding GFP-fusion mutants. Surprisingly, the N-terminal construct showed only cytosolic localization, whereas the C-terminal construct localized exclusively to the centrosome. This is unexpected because Stu2p is localized at the spindle pole body. Full-length DdCP224-GFP was present both at centrosomes and along microtubules. Furthermore, it bound to microtubules in vitro, unlike the two truncated mutants. Thus centrosome binding is determined by the C-terminal half and microtubule binding may require the interaction of the N- and C-terminal halves. Interestingly, cells expressing full-length DdCP224-GFP exhibit supernumerary centrosomes and show a cytokinesis defect, suggesting that DdCP224 plays an important role in centrosome duplication. These features are unique among the known centrosomal proteins.
A β-catenin/engrailed chimera selectively suppresses Wnt signaling
Authors: Montross Werner T. , Ji Hong , McCrea Pierre D.
Published: April 2021

β-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, β-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal ‘organizer’ region of early Xenopus embryos, β-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that β-catenin plays in Wnt signaling, we generated a chimeric protein, β-Engrailed (β-Eng), in which the C-terminal trans-activation domain of β-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, β-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress β-catenin’s signaling function apart from its role in cadherin-mediated cell-cell adhesion.
Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?
Authors: Petrone Angiola , Sap Jan
Published: April 2021

Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity.
Contraction of collagen matrices mediated by α2β1A and αvβ3 integrins
Authors: Cooke Mary E. , Sakai Takao , Mosher Deane F.
Published: April 2021

The β1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of β1 and β3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the β1A splice variant of β1 (β1A-GD25) did not express α2β1A and did not adhere to collagen. After transfection of α2 into β1A-GD25 cells, the α2β1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas β1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to αvβ3 or a RGD-containing peptide, indicating that αvβ3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by α2β1A-GD25 cells was not inhibited by antibodies to αvβ3 or RGD-containing peptide, but was inhibited by anti-α2 antibody. Flow cytometry demonstrated negligible expression of αvβ3 by β1A-GD25 and α2β1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by α2β1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by α2β1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the β1-null GD25 cells and β1A and α2β1A transfectants demonstrate that α2β1A and αvβ3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.
Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis
Authors: Wong Kelvin C. Y. , Naqvi Naweed I. , Iino Yuichi , Yamamoto Masayuki , Balasubramanian Mohan K.
Published: April 2021

Cell division in many eukaryotes, including the fission yeast Schizosaccharomyces pombe, utilizes a contractile actomyosin ring. In S. pombe, the actomyosin ring is assembled at the medial cortex upon entry into mitosis and constricts at the end of anaphase to guide the centripetal deposition of the septum. Despite identification of several structural components essential for actomyosin ring assembly, the interdependencies between these geneproducts in the process of ring assembly are unknown. This study investigates the role of Rng3p, a member of the UCS-domain containing protein family (Unc-45p, Cro1p, She4p), in actomyosin ring assembly. Null mutants in rng3 resemble deletion mutants in the type II myosin heavy chain (myo2) and rng3ts mutants show strong negative interactions with the myo2-E1 mutant, suggesting that Rng3p is involved in modulating aspects of type II myosin function. Interestingly, a green fluorescent protein (GFP) tagged Rng3p fusion is detected at the division site in the myo2- E1 mutant, but not in other myo2-alleles, wildtype cells or in 18 other cytokinesis mutants. Assembly and maintenance of Rng3p at the division site in the myo2- E1 mutant requires F-actin. Rng3p is also required for the proper assembly of Myo2p and F-actin into a functional actomyosin ring but is not necessary for their accumulation at the division site. We conclude that Rng3p is a novel component of the F-actin cytoskeleton essential for a late step in actomyosin ring assembly and that it might monitor some aspect of type II myosin assembly during actomyosin ring construction.
Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis
Authors: Hofmann Ilse , Mertens Claudia , Brettel Monika , Nimmrich Volker , Schnölzer Martina , Herrmann Harald
Published: April 2021

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phasebinding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several μm. The basic aminoterminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by coimmunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound – and does not bind – to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.
XCS-1, a maternally expressed gene product involved in regulating mitosis in Xenopus
Authors: Nakamura Hisashi , Wu Chuanfen , Kuang Jian , Larabell Carolyn , Etkin Laurence D.
Published: April 2021

The regulation of the cell cycle during early development is an important and complex biological process. We have cloned a cDNA, XCS-1, that may play an important role in regulating mitosis during early embryogenesis in Xenopus laevis. XCS-1 is a maternally expressed gene product that is the Xenopus homologue of the human cleavage signal protein (CS-1). XCS-1 transcripts were detected in oocytes with the titer decreasing just prior to the MBT. During development the XCS-1 protein was detected on the membrane and in the nucleus of blastomeres. It was also detected on the mitotic spindle in mitotic cells and on the centrosomes in interphase cells. Overexpression of myc-XCS-1 in Xenopus embryos resulted in abnormal mitoses with increased numbers of centrosomes, multipolar spindles, and abnormal distribution of chromosomes. Also, we observed incomplete cytokinesis resulting in multiple nuclei residing in the same cytoplasm with the daughter nuclei in different phases of the cell cycle. The phenotype depended on the presence of the N terminus of XCS-1 (aa 1-73) and a consensus NIMA kinase phosphorylation site (aa159-167). Mutations in this site affected the ability of the overexpressed XCS-1 protein to produce the phenotype. These results suggest that XCS-1 is a maternal factor playing an important role in the regulation of the cell cycle during early embryogenesis and that its function depends on its state of phosphorylation.
Coupled transport of p24 family members
Authors: Emery Gregory , Rojo Manuel , Gruenberg Jean
Published: April 2021

Recent studies show that small transmembrane proteins of ≈22-24 kDa (the p24 family), which are grouped into 4 subfamilies by sequence homology (p23, p24, p25 and p26), are involved in the early secretory pathway. In this study, we have investigated the mutual requirements of ectopically expressed members of the p24 family for targeting to their proper cellular destination. We find that coexpression of p23 and p24 is both necessary and sufficient for each protein to be transported to the cis-Golgi network/Golgi complex. Proteins from other subfamilies did not substitute for either p23 or p24, even after multiple coexpression. However, trafficking of the p23/p24 couple was facilitated by coexpression of proteins from other subfamilies. In addition, we find that the sequence resembling an endoplasmic reticulum retrieval signal present in the cytoplasmic domain of p23 (but not p24) is dispensable. In contrast, the conserved coiledcoil region in the lumenal domain is absolutely required in both p23 and p24 for proper targeting of the p23/p24 couple. These data demonstrate that p23 and p24 must interact with each other to reach their destination, but that this strict requirement is combined with a mutual dependence amongst p24 proteins. We speculate that p24 proteins can form different oligomeric complexes, which contribute to confer specialized sorting/trafficking properties to membranes of the early secretory pathway, perhaps serving as membrane organizers.
AAA domains and organization of the dynein motor unit
Authors: King Stephen M.
Published: April 2021

Dyneins contain one-three microtubule motor units that are each derived from the C-terminal globular head of a heavy chain. The N-terminal regions of the heavy chains form stems that are required for intra-dynein associations. The microtubule-binding sites are located at the terminus of a short stalk that emanates from each globular head. Recent electron microscopic analysis indicates that the dynein head has a heptameric toroidal organization. This finding is echoed by the identification of six AAA (ATPases associated with cellular activities) domains and a seventh unrelated unit within this heavy chain region. At least two of these AAA domains can bind nucleotide, although only one appears able to hydrolyze ATP. Several other AAA domain proteins exhibit a similar annular organization of six AAA units. Detailed structural information is available for several AAA proteins, including N-ethylmaleimidesensitive vesicle-fusion protein and the RuvB motor involved in DNA migration and resolution of Holliday junctions. The resulting structural parallels allow intriguing predictions to be made concerning dynein organization and motor function.
RecQ-like helicases: the DNA replication checkpoint connection
Authors: Frei Christian , Gasser Susan M.
Published: April 2021

The eukaryotic homologues of the Escherichia coli RecQ DNA helicase play conserved roles in the maintenance of genome stability. Results obtained in yeast and mammalian systems are beginning to form a coherent picture about what these helicases do to ensure normal cell division and why humans who lack these enzymes are cancer prone. Recent data suggest that the yeast enzyme Sgs1p, as well as two human homologues, which are encoded by the Bloom’s and Werner’s syndrome genes, function during DNA replication and possibly in a replication checkpoint specific to S phase.
Assembly of multiple dystrobrevin-containing complexes in the kidney
Authors: Loh Nellie Y. , Newey Sarah E. , Davies Kay E. , Blake Derek J.
Published: April 2021

Dystrophin is the key component in the assembly and maintenance of the dystrophin-associated protein complex (DPC) in skeletal muscle. In kidney, dystroglycan, an integral component of the DPC, is involved in kidney epithelial morphogenesis, suggesting that the DPC is important in linking the extracellular matrix to the internal cytoskeleton of kidney epithelia. Here, we have investigated the molecular architecture of dystrophin-like protein complexes in kidneys from normal and dystrophin- deficient mice. Using isoform-specific antibodies, we show that the different cell types that make up the kidney maintain different dystrophin-like complexes. These complexes can be broadly grouped according to their dystrobrevin content: β-dystrobrevin containing complexes are present at the basal region of renal epithelial cells, whilst α-dystrobrevin-1 containing complexes are found in endothelial and smooth muscle cells. Furthermore, these complexes are maintained even in the absence of all dystrophin isoforms. Thus our data suggest that the functions and assembly of the dystrophin-like complexes in kidney differ from those in skeletal muscle and implicate a protein other than dystrophin as the primary molecule in the assembly and maintenance of kidney complexes. Our findings also provide a possible explanation for the lack of kidney pathology in Duchenne muscular dystrophy patients and mice lacking all dystrophin isoforms.
Amino-terminal domain of classic cadherins determines the specificity of the adhesive interactions
Authors: Klingelhöfer Jörg , Troyanovsky Regina B. , Laur Oscar Y. , Troyanovsky Sergey
Published: April 2021

Classic cadherins are transmembrane receptors involved in cell type-specific calcium-dependent intercellular adhesion. The specificity of adhesion is mediated by homophilic interactions between cadherins extending from opposing cell surfaces. In addition, classic cadherins can self- associate forming lateral dimers. Whereas it is widely excepted that lateral dimerization of cadherins is critical for adhesion, details of this process are not known. Yet, no evidence for physical association between different classic cadherins in cells expressing complex cadherin patterns has been reported. To study lateral and adhesive intercadherin interactions, we examined interactions between two classic cadherins, E- and P-cadherins, in epithelial A-431 cells co- producing both proteins. We showed that these cells exhibited heterocomplexes consisting of laterally assembled E- and P-cadherins. These complexes were formed by a mechanism involving Trp156 of E-cadherin. Removal of calcium ions from the culture medium triggered a novel Trp156-independent type of lateral E-cadherin-P-cadherin association. Notably, an antiparallel (adhesive) mode of interaction between these cadherins was negligible. The specificity of adhesive interaction was localized to the amino-terminal (EC1) domain of both cadherins. Thus, EC1 domain of classic cadherins exposes two determinants responsible for nonspecific lateral and cadherin type- specific adhesive dimerization.
The chemotactic and mitogenic effects of platelet-derived growth factor-BB on rat aorta smooth muscle cells are inhibited by basic fibroblast growth factor
Authors: Facchiano Antonio , Marchis Francesco De , Turchetti Elisabetta , Facchiano Francesco , Guglielmi Mario , Denaro Alessandra , Palumbo Roberta , Scoccianti Marco , Capogrossi Maurizio C.
Published: April 2021

In response to endovascular injury, platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) are released locally and modulate vascular smooth muscle cells (SMC) proliferation and migration within the vascular wall. The aim of the present in vitro study was to determine how rat aorta SMC respond to the simultaneous exposure to PDGF-BB and bFGF. In a modified Boyden chamber assay bFGF exhibited a dose-dependent effect to inhibit the chemotactic action of PDGF-BB. A comparable result was observed in proliferation assays. In contrast, MIP-1 β, epidermal growth factor (EGF), fibronectin and acidic FGF (aFGF) did not inhibit the chemotactic effect of PDGF-BB. Denatured bFGF did not exert an inhibitory effect and neutralizing antibodies either to bFGF or to bFGF-receptor abolished the inhibition observed in the presence of bFGF. The role played by PDGF receptor α (PDGF-Rα) was investigated in PDGF-Rα-dominant negative-transfected SMC, by selectively blocking PDGF- BB-binding to PDGF-Rα with neomycin, by neutralizing PDGF-Rα with a monoclonal antibody and by selectively stimulating PDGF-Rα with PDGF-AA; in all cases the effect of bFGF to inhibit PDGF-BB-directed SMC migration was abolished.

These in vitro studies show that bFGF significantly inhibits PDGF-BB-induced SMC migration and proliferation and that this effect is mediated by both PDGF-Rα and bFGF receptor.
Tension on chromosomes increases the number of kinetochore microtubules but only within limits
Authors: King Jennifer M. , Nicklas R. Bruce
Published: April 2021

When chromosomes attach properly to a mitotic spindle, their kinetochores generate force in opposite directions, creating tension. Tension is presumed to increase kinetochore microtubule number, but there has been no direct evidence this is true. We micromanipulated grasshopper spermatocyte chromosomes to test this assumption and found that tension does indeed affect the number of kinetochore microtubules. Releasing tension at kinetochores causes a drop to less than half the original number of kinetochore microtubules. Restoring tension onto these depleted kinetochores restores the microtubules to their original number. However, the effects of tension are limited. Prometaphase kinetochores, when under normal tension from mitotic forces, have about half as many microtubules as they will in late metaphase. We imposed a tension force of 6×10−5 dynes, three times the normal tension, on prometaphase kinetochores. The elevated tension did not drive kinetochore microtubule number above normal prometaphase values. Tension probably increases the number of kinetochore microtubules by slowing their turnover rate. The limited effect of tension at prometaphase kinetochores suggests that they have fewer microtubule binding sites than at late metaphase. The relatively few sites available in prometaphase may be the decisive sites whose binding of microtubules regulates the dynamics of transient kinetochore constituents, including checkpoint components.
Antisense oligonucleotide experiments elucidate the essential role of titin in sarcomerogenesis in adult rat cardiomyocytes in long-term culture
Authors: Person Veronika , Kostin Sawa , Suzuki Keisuke , Labeit Siegfried , Schaper Jutta
Published: April 2021

An essential role of titin as a molecular ruler in sarcomerogenesis has been frequently discussed. In this study, we tested the hypothesis that the expression of titin is a prerequisite for thick filament incorporation into sarcomeres by using an antisense oligonucleotide approach to interfere with titin translation in the de-/ redifferentiation model of adult rat cardiomyocytes (ARC) in long-term culture. As a first step, the growth pattern ranging from rod shape to round and later to spreading cells and the cell surface area of ARC were quantitatively evaluated and standardized. This represents the basis for experiments interfering with sarcomere formation using three different antisense phosphorothioate oligonucleotides (S-ODN) at a dosage of 10 μM specific for titin mRNA. Presence of fluorescein labeled S-ODN in ARC indicated cellular uptake and both, antisense and random S-ODN, induced a significant increase in cell size as compared with control untreated ARC. At days 12 and 16 in culture, antisense S-ODN treatment resulted in reduced expression of titin and disturbance of myosin incorporation into sarcomeres, evident by diffuse myosin labeling and a significantly decreased area of regular myosin cross-striation (control 75%, day 12 S-ODN 20%, day 16 14%) shown by laser scanning confocal microscopy. Cellular integrity indicated by presence of α-actinin was not disturbed. These findings provide evidence for the role of titin as a template for myosin incorporation and therefore as a prerequisite for sarcomerogenesis.
Intact Ca2+-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells
Authors: Rescher Ursula , Zobiack Nicole , Gerke Volker
Published: April 2021

Annexin 1 is a Ca2+-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells. To establish its localization in live cells, we followed the intracellular fate of annexin 1 molecules fused to the Green Fluorescent Protein (GFP). We show that annexin 1-GFP associates with distinct, transferrin receptor-positive membrane structures in living HeLa cells. A GFP chimera containing the Ca2+/phospholipid-binding protein core of annexin 1 also shows a punctate intracellular distribution, although the structures labeled here do not resemble early but, at least in part, late endosomes. In contrast, the cores of annexins 2 and 4 fused to GFP exhibit a cytoplasmic or a different punctate distribution, respectively, indicating that the highly homologous annexin core domains carry distinct membrane specificities within live cells. By inactivating the three high-affinity Ca2+ binding sites in annexin 1 we also show that endosomal membrane binding of the protein in live HeLa cells depends on the integrity of these Ca2+ binding sites. More detailed analysis identifies a single Ca2+ site in the second annexin repeat that is crucially involved in establishing the membrane association. These results reveal for the first time that intracellular membrane binding of an annexin in living cells requires Ca2+ and is mediated in part through an annexin core domain that is capable of establishing specific interactions.
Fast transport of neurofilament protein along microtubules in squid axoplasm
Authors: Prahlad Veena , Helfand Brian T. , Langford George M. , Vale Ron D. , Goldman Robert D.
Published: April 2021

Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 μm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 μm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal proteins, throughout neurons.
Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells
Authors: Yan Li , Moses Marsha A. , Huang Sui , Ingber Donald E.
Published: April 2021

The growth and regression of capillary blood vessels during angiogenesis is greatly influenced by changes in the activity of matrix metalloproteinases (MMPs), which selectively degrade extracellular matrix (ECM) and thereby modulate capillary endothelial cell shape, growth and viability. However, changes in cell-ECM binding and cell spreading have also been reported to alter MMP secretion and activation. Studies were carried out to determine whether changes in integrin binding or cell shape feed back to alter MMP-2 processing in human capillary endothelial (HCE) cells. Catalytic processing of proMMP-2 to active MMP-2 progressively decreased when HCE cells were cultured on dishes coated with increasing densities of fibronectin (FN), which promote both integrin binding and cell spreading. Conversely, the highest levels of active MMP-2 were detected in round cells cultured on low FN. When measured 24 hours after plating, this increase in active MMP-2 was accompanied by a concomitant rise in mRNA and protein levels for the membrane-type 1 MMP (MT1-MMP), which catalyzes the cleavage of proMMP-2. To determine whether proMMP-2 processing was controlled directly by integrin binding or indirectly by associated changes in cell shape, round cells on low FN were allowed to bind to microbeads (4.5 μm diameter) coated with a synthetic RGD peptide or FN; these induce local integrin receptor clustering without altering cell shape. ProMMP-2 activation was significantly decreased within minutes after bead binding in these round cells, prior to any detectable changes in expression of MT1-MMP, whereas binding of beads coated with control ligands for other transmembrane receptors had no effect. This inhibitory effect was mimicked by microbeads coated with activating antibodies against αVβ3 and β1 integrins, suggesting a direct role for these cell-surface ECM receptors in modulating proMMP-2 activation. Similar inhibition of proMMP-2 processing by integrin binding, independent of cell spreading, was demonstrated in cells that were cultured on small, microfabricated adhesive islands that prevented cell spreading while presenting a high FN density directly beneath the cell. Interestingly, when spread cells were induced to round up from within by disrupting their actin cytoskeleton using cytochalasin D, proMMP-2 processing did not change at early times; however, increases in MT1-MMP mRNA levels and MMP-2 activation could be detected by 18 hours. Taken together, these results suggest the existence of two phases of MMP-2 regulation in HCE cells when they adhere to ECM: (1) a quick response, in which integrin clustering alone is sufficient to rapidly inhibit processing of proMMP-2 and (2) a slower response, in which subsequent cell spreading and changes in the actin cytoskeleton feed back to decrease expression of MT1-MMP mRNA and, thereby, further suppress cellular proteolytic activity.
The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1
Authors: Bryan Janine T. , Morasso Maria I.
Published: April 2021

The murine Dlx3 protein is a putative transcriptional activator that has been implicated during development and differentiation of epithelial tissue. Dlx3 contains a homeodomain and mutational analysis has revealed two regions, one N-terminal and one C-terminal to the homeodomain, that act as transcriptional activators in a yeast one-hybrid assay. In addition to transactivation, data are presented to demonstrate specific DNA binding and an association between Dlx3 and the Msx1 protein in vitro. Immunohistochemical analysis confirmed coexpression of Dlx3 and Msx1 proteins in the differentiated layers of murine epidermal tissues.

Transcription factor function requires nuclear localization. In this study, the intracellular localization of the green fluorescent protein fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium and is shown to localize to the nucleus. A bipartite nuclear localization signal (NLS) was identified by mutational analysis and shown to be sufficient for nuclear localization. This was demonstrated by insertion of the Dlx3 bipartite NLS sequence into a cytoplasmic fusion protein, GFP-keratin 14, which functionally redirected GFP-keratin 14 expression to the nucleus. Further analysis of Dlx3 NLS mutants revealed that the Dlx3 NLS sequences are required for specific DNA binding, transactivation potential and interactions with the Msx1 protein.
SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands
Authors: Niemeyer Barbara A. , Schwarz Thomas L.
Published: April 2021

Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. Here we report the identification and characterization of SNAP-24, a closely related Drosophila SNAP-25 homologue, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion.
Defective organellar membrane protein trafficking in Ap3b1-deficient cells
Authors: Yang Wei , Li Chaoying , Ward Diane M. , Kaplan Jerry , Mansour Suzanne L.
Published: April 2021

AP-3 is a heterotetrameric protein complex involved in intracellular vesicle transport. Molecular analyses show that Ap3b1, which encodes the AP-3 β3A subunit, is altered in pearl mice. To provide genetic evidence that mutation of Ap3b1 is responsible for the pearl phenotype and to determine the null phenotype, the Ap3b1 gene was disrupted by homologous recombination. Mice homozygous for the resulting allele, Ap3b1LN, or compound heterozygotes with pearl, displayed phenotypes similar to those of pearl mice, confirming that Ap3b1 is the causal gene for pearl. Moreover, pearl is likely to be a hypomorph as the Ap3b1LN homozygotes had a lighter coat color and accumulated fewer of the μ3 and δ3 subunits of AP-3 than did pearl mice. Finally, immunofluorescence analysis of fibroblasts and melanocytes cultured from Ap3b1LN homozygotes revealed that the lysosomal membrane proteins Lamp I and Lamp II and the melanosomal membrane protein tyrosinase were mislocalized. In particular, the Lamp proteins were clustered on the cell surface. These findings strengthen the evidence for an alternate pathway via the plasma membrane for cargo normally transported to organelles by AP-3.