bluuebunny/crossref_metadata_2025 huggingface dataset:
The organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons.
Authors: Krijnse-Locker J , Parton R G , Fuller S D , Griffiths G , Dotti C G
Published: August 2013

The boundaries of the organelles of the biosynthetic endomembrane system are still controversial. In this paper we take advantage of the unique architectural organization of neurons to investigate the localization of a spectrum of compartment-specific markers with the goal of defining the location of the rough endoplasmic reticulum (ER), smooth ER, intermediate compartment, and the Golgi complex. Markers of the rough ER (signal sequence receptor), Golgi complex (mannosidase II), and the trans Golgi network (TGN38) were essentially restricted to the cell body and the initial segment of one of the cell's dendrites. In contrast the cytochemical reaction product for glucose 6 phosphate, a classical ER marker, in addition to staining ER structures in the cell body also reacted with smooth ER elements that extended into both axons and dendrites. These peripheral smooth ER elements also reacted at the immunofluorescence level for ER marker 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as for calnexin and protein disulfide isomerase. We also analyzed the location of rab1, rab2, p58, the KDEL receptor, and beta-subunit of coatomer. These intermediate compartment markers were found predominantly in the cell body but also extended to the proximal parts of the dendrites. Collectively, our data argue that the ER of hippocampal neurons consists of functionally and spatially distinct and separated domains, and they stress the power of the hippocampal neuron system for investigations of the organization of the ER by light microscopy.
Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro.
Authors: Campbell S D , Sprenger F , Edgar B A , O'Farrell P H
Published: August 2013

Cdc2 kinase activity is required for triggering entry into mitosis in all known eukaryotes. Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, we screened for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homologue. Dwee1 kinase is closely related to human and Xenopus Wee1 homologues, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast.
Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.
Authors: Plopper G E , McNamee H P , Dike L E , Bojanowski K , Ingber D E
Published: August 2013

Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60c-src, pp125FAK, phosphatidylinositol-3-kinase, phospholipase C-gamma, and Na+/H+ antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp60c-src, pp125FAK, phospholipase C-gamma, and the Na+/H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60c-src and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.
Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae.
Authors: Smith M G , Simon V R , O'Sullivan H , Pon L A
Published: August 2013

During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast.
Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells.
Authors: Preuss U , Döring F , Illenberger S , Mandelkow E M
Published: August 2013

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.
Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.
Authors: Guan T , Müller S , Klier G , Panté N , Blevitt J M , Haner M , Paschal B , Aebi U , Gerace L
Published: August 2013

The p62 complex is an oligomeric assembly of O-linked glycoproteins of the nuclear pore complex that interacts with cytosolic transport factors and is part of the machinery for nuclear protein import. In this study we have purified the p62 complex from rat liver nuclear envelopes and analyzed its structure and composition. The p62 complex consists of four distinct polypeptides (p62, p58, p54, and p45) and has a mass of approximately 234 kDa, calculated from its hydrodynamic properties and supported by chemical cross-linking and scanning transmission electron microscopy. These data suggest that the p62 complex contains one copy of each constituent polypeptide. Analysis of preparations of the p62 complex by electron microscopy using rotary metal shadowing and negative staining revealed donut-shaped particles with a diameter of approximately 15 nm. Immunogold electron microscopy of isolated rat liver nuclear envelopes demonstrated that p62 occurs on both the nucleoplasmic and cytoplasmic sides of the pore complex near the central gated channel involved in active transport of proteins and RNAs. The properties and localization of the p62 complex suggest that it may be involved in binding transport ligands near the center of the nuclear pore complex and in subsequently transferring them to the gated transport channel.
The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.
Authors: Steinberg G , Schliwa M
Published: August 2013

The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins.
Force Generation by Microtubule Assembly/Disassembly in Mitosis and Related Movements
Authors: Inoué Shinya , Salmon Edward D.
Published: August 2013

In this article, we review the dynamic nature of the filaments (microtubules) that make up the labile fibers of the mitotic spindle and asters, we discuss the roles that assembly and disassembly of microtubules play in mitosis, and we consider how such assembling and disassembling polymer filaments can generate forces that are utilized by the living cell in mitosis and related movements.
Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.
Authors: DiNubile M J , Cassimeris L , Joyce M , Zigmond S H
Published: August 2013

A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution.
Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.
Authors: Kwak B R , Hermans M M , De Jonge H R , Lohmann S M , Jongsma H J , Chanson M
Published: August 2013

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.
A mating type-linked mutation that disrupts the uniparental inheritance of chloroplast DNA also disrupts cell-size control in Chlamydomonas.
Authors: Armbrust E V , Ibrahim A , Goodenough U W
Published: August 2013

An intriguing feature of early zygote development in Chlamydomonas reinhardtii is the active elimination of chloroplast DNA from the mating-type minus parent due presumably to the action of a zygote-specific nuclease. Meiotic progeny thus inherit chloroplast DNA almost exclusively from the mating-type plus parent. The plus-linked nuclear mutation mat3 prevents this selective destruction of minus chloroplast DNA and generates progeny that display a biparental inheritance pattern. Here we show that the mat3 mutation creates additional phenotypes not previously described: the cells are much smaller than wild type and they possess substantially reduced amounts of both mitochondrial and chloroplast DNA. We propose that the primary defect of the mat3 mutation is a disruption of cell-size control and that the inhibition of the uniparental transmission of chloroplast genomes is a secondary consequence of the reduced amount of chloroplast DNA in the mat3 parent.
Generation of truncated forms of the NG2 proteoglycan by cell surface proteolysis.
Authors: Nishiyama A , Lin X H , Stallcup W B
Published: August 2013

NG2 is a chondroitin sulfate proteoglycan that is expressed on dividing progenitor cells of several lineages including glia, muscle, and cartilage. It is an integral membrane proteoglycan with a core glycoprotein of 300 kDa. In the present study we have characterized three molecular forms of the NG2 core protein expressed by different cell lines. Many cell lines that express the full length 300-kDa NG2 core protein also release a 290-kDa form into the medium. This species lacks the cytoplasmic domain but contains almost the entire ectodomain. Two core protein species, the intact 300-kDa form and a truncated 275-kDa form, are expressed at the surface of an NG2-transfected cell line U251NG52. The 275-kDa species lacks the cytoplasmic domain and at least 64 amino acids of the ectodomain. Mild trypsinization of B49 cells also generates the 275-kDa species, suggesting that this component is produced by proteolysis of the 300-kDa form. Conversion of the 300-kDa species to the 275-kDa form in U251NG52 cells is stimulated by reagents such as phorbol esters, which activate protein kinase C. Phorbol esters are also known to induce expression of metalloproteinases such as collagenase and stromelysin, which could be responsible for cleavage of the 300-kDa core protein. Although B49 cells do not spontaneously produce the truncated 275-kDa species, use of monoclonal antibodies against NG2 to block the interaction between NG2 and type VI collagen results in the appearance of the 275-kDa component in these cells. Thus the interaction between NG2 and type VI collagen, which contains a Kunitz-type proteinase inhibitor sequence in the alpha 3 chain, may protect the proteoglycan against proteolysis. This is consistent with the observed deficiency of U251NG52 cells in anchoring type VI collagen at the surface.
Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.
Authors: Coffin J D , Florkiewicz R Z , Neumann J , Mort-Hopkins T , Dorn G W , Lightfoot P , German R , Howles P N , Kier A , O'Toole B A
Published: August 2013

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.
Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.
Authors: Lewis J M , Schwartz M A
Published: August 2013

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.
Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.
Authors: Izumi T , Maller J L
Published: August 2013

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.
NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration.
Authors: Xiang X , Osmani A H , Osmani S A , Xin M , Morris N R
Published: August 2013

During a study of the genetics of nuclear migration in the filamentous fungus Aspergillus nidulans, we cloned a gene, nudF, which is required for nuclear migration during vegetative growth as well as development. The NUDF protein level is controlled by another protein NUDC, and extra copies of the nudF gene can suppress the nudC3 mutation. nudF encodes a protein with 42% sequence identity to the human LIS-1 (Miller-Dieker lissencephaly-1) gene, which is required for proper neuronal migration during brain development. This strong similarity suggests that the LIS-1 gene product may have a function similar to that of NUDF and supports previous findings to suggest that nuclear migration may play a role in neuronal migration.
Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.
Authors: Iruela-Arispe M L , Lane T F , Redmond D , Reilly M , Bolender R P , Kavanagh T J , Sage E H
Published: August 2013

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.
Inhibition of cyclin-dependent kinases by p21.
Authors: Harper J W , Elledge S J , Keyomarsi K , Dynlacht B , Tsai L H , Zhang P , Dobrowolski S , Bai C , Connell-Crowley L , Swindell E
Published: August 2013

p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.
Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes.
Authors: Li O , Heath C V , Amberg D C , Dockendorff T C , Copeland C S , Snyder M , Cole C N
Published: August 2013

To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)50 probe, we identified those that showed nuclear accumulation of poly(A)+ RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT-ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37 degrees C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)+ RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.
The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.
Authors: Bovia F , Fornallaz M , Leffers H , Strub K
Published: August 2013

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.
Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.
Authors: Grady E F , Garland A M , Gamp P D , Lovett M , Payan D G , Bunnett N W
Published: August 2013

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.
Polarized distribution of actin isoforms in gastric parietal cells.
Authors: Yao X , Chaponnier C , Gabbiani G , Forte J G
Published: August 2013

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.
Loss of cell surface syndecan-1 causes epithelia to transform into anchorage-independent mesenchyme-like cells.
Authors: Kato M , Saunders S , Nguyen H , Bernfield M
Published: August 2013

Simple epithelial cells are polygonal in shape, polarized in an apical-basal orientation, and organized into closely adherent sheets, characteristics that result from a variety of cellular specializations and adhesive proteins. These characteristics are lost when the epithelia transform during embryogenesis into mesenchymal cells or after neoplasia into invasive carcinoma cells. Of the syndecan family of transmembrane heparan sulfate proteoglycans, simple epithelia produce predominantly syndecan-1, which is found at basolateral surfaces and within adhesive junctions. To elucidate the function of this syndecan-1, normal murine mammary gland epithelia were made deficient in syndecan-1 by transfection with an expression vector containing the syndecan-1 cDNA in the antisense configuration. Several independently derived clones of stable transfectants contained the antisense cDNA in their genome and expressed the antisense transcript. These grew either as epithelial islands of closely adherent polygonal cells, identical to both the parental cells and the vector-only control transfectants, or as individual elongated fusiform cells that invaded and migrated within collagen gels, like mesenchymal cells, but were anchorage-independent for growth. The clones that retained epithelial characteristics were moderately deficient in cell surface syndecan-1 (greater than 48% of control levels) but did not differ from control cells in expression of beta 1-integrins and E-cadherin, or in F-actin organization. However, the clones of fusiform cells were severely deficient in cell surface syndecan-1 (less than 12% of control levels) and showed rearranged beta 1-integrins, markedly reduced E-cadherin expression, and disorganized F-actin filaments, but retained mammary epithelial markers. Therefore, depleting epithelia of cell surface syndecan-1 alters cell morphology and organization, the arrangement and expression of adhesion molecules, and anchorage-dependent growth controls. Thus, cell surface syndecan-1 is required to maintain the normal phenotype of simple epithelia.
Specialized functional properties of the integrin alpha 4 cytoplasmic domain.
Authors: Kassner P D , Alon R , Springer T A , Hemler M E
Published: August 2013

For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.
The light chain p28 associates with a subset of inner dynein arm heavy chains in Chlamydomonas axonemes.
Authors: LeDizet M , Piperno G
Published: August 2013

We show here that I2 and I3 inner dynein arm heavy chains of Chlamydomonas axonemes are resolved into two classes: one class associated with the protein p28 and the other associated with the protein caltractin/centrin. We have determined the nucleotide sequence of the gene encoding p28, a light chain that, together with actin and caltractin/centrin, is associated with inner dynein arms I2 and I3 of Chlamydomonas axonemes. p28 is a novel protein with affinity for a subset of the inner dynein arm heavy chains, but with no apparent significant homologies to tubulin- or actin-binding proteins. An antiserum specific for p28 showed that p28 is present along the entire axoneme. The same antiserum coimmunoprecipitated p28, actin, and dynein heavy chains 2' and 2. In contrast, an anti-caltractin/centrin antiserum coimmunoprecipitated caltractin/centrin, actin, and the heavy chains 2, 3, and 3'. It is likely that the dynein heavy chain 2 associated with p28, referred to as 2A, is a different polypeptide from dynein heavy chain 2 bound to caltractin/centrin, referred to as 2B. The complex formed by heavy chain 2B, actin, and caltractin/centrin is preferentially extracted by exposure to Nonidet P-40 and is missing in mutants lacking components 1 and 2 of the dynein regulatory complex.
The origin recognition complex in silencing, cell cycle progression, and DNA replication.
Authors: Loo S , Fox C A , Rine J , Kobayashi R , Stillman B , Bell S
Published: August 2013

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.
The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex.
Authors: Dingwall A K , Beek S J , McCallum C M , Tamkun J W , Kalpana G V , Goff S P , Scott M P
Published: August 2013

During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin.