bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Two RNA-binding Proteins Associated with Human Telomerase RNA
Authors: Le Siyuan , Sternglanz Rolf , Greider Carol W.
Published: July 2013

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.
A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61
Authors: Zirwes Rudolf F. , Eilbracht Jens , Kneissel Sandra , Schmidt-Zachmann Marion S.
Published: July 2013

We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of “DEAD-box” proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ∼11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ∼4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.
Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons
Authors: Kaether Christoph , Skehel Paul , Dotti Carlos G.
Published: July 2013

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.
The PC6B Cytoplasmic Domain Contains Two Acidic Clusters That Direct Sorting to Distincttrans-Golgi Network/Endosomal Compartments
Authors: Xiang Yang , Molloy Sean S. , Thomas Laurel , Thomas Gary
Published: July 2013

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A–dispersible, BaCl2-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr1802GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.
A New Paxillin-binding Protein, PAG3/Papα/KIAA0400, Bearing an ADP-Ribosylation Factor GTPase-activating Protein Activity, Is Involved in Paxillin Recruitment to Focal Adhesions and Cell Migration
Authors: Kondo Akiko , Hashimoto Shigeru , Yano Hajime , Nagayama Kuniaki , Mazaki Yuichi , Sabe Hisataka
Published: July 2013

Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, PAG3 (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. PAG3 bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Papα, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of PAG3 with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of PAG3. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities.
Clonal Tests of Conventional Kinesin Function during Cell Proliferation and Differentiation
Authors: Brendza Robert P. , Sheehan Kathy B. , Turner F.R. , Saxton William M.
Published: July 2013

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.
Intracellular and Extracellular Leukemia Inhibitory Factor Proteins Have Different Cellular Activities That Are Mediated by Distinct Protein Motifs
Authors: Haines Bryan P. , Voyle Roger B. , Rathjen Peter D.
Published: July 2013

Although many growth factors and cytokines have been shown to be localized within the cell and nucleus, the mechanism by which these molecules elicit a biological response is not well understood. The cytokine leukemia inhibitory factor (LIF) provides a tractable experimental system to investigate this problem, because translation of alternatively spliced transcripts results in the production of differentially localized LIF proteins, one secreted from the cell and acting via cell surface receptors and the other localized within the cell. We have used overexpression analysis to demonstrate that extracellular and intracellular LIF proteins can have distinct cellular activities. Intracellular LIF protein is localized to both nucleus and cytoplasm and when overexpressed induces apoptosis that is inhibited by CrmA but not Bcl-2 expression. Mutational analysis revealed that the intracellular activity was independent of receptor interaction and activation and reliant on a conserved leucine-rich motif that was not required for activation of cell surface receptors by extracellular protein. This provides the first report of alternate intracellular and extracellular cytokine activities that result from differential cellular localization of the protein and are mediated by spatially distinct motifs.
A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization
Authors: Hettmann Christine , Herm Angelika , Geiter Ariane , Frank Bernd , Schwarz Eva , Soldati Thierry , Soldati Dominique
Published: July 2013

Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.
ADAM 23/MDC3, a Human Disintegrin That Promotes Cell Adhesion via Interaction with the αvβ3 Integrin through an RGD-independent Mechanism
Authors: Cal Santiago , Freije José M.P. , López José M. , Takada Yoshikazu , López-Otı́n Carlos
Published: July 2013

ADAM 23 (a disintegrin and metalloproteinase domain)/MDC3 (metalloprotease, disintegrin, and cysteine-rich domain) is a member of the disintegrin family of proteins expressed in fetal and adult brain. In this work we show that the disintegrin-like domain of ADAM 23 produced in Escherichia coli and immobilized on culture dishes promotes attachment of different human cells of neural origin, such as neuroblastoma cells (NB100 and SH-Sy5y) or astrocytoma cells (U373 and U87 MG). Analysis of ADAM 23 binding to integrins revealed a specific interaction with αvβ3, mediated by a short amino acid sequence present in its putative disintegrin loop. This sequence lacks any RGD motif, which is a common structural determinant supporting αvβ3-mediated interactions of diverse proteins, including other disintegrins. αvβ3 also supported adhesion of HeLa cells transfected with a full-length cDNA for ADAM 23, extending the results obtained with the recombinant protein containing the disintegrin domain of ADAM 23. On the basis of these results, we propose that ADAM 23, through its disintegrin-like domain, may function as an adhesion molecule involved in αvβ3-mediated cell interactions occurring in normal and pathological processes, including progression of malignant tumors from neural origin.
A Trypanosomal Protein Synergizes with the Cytokines Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor to Prevent Apoptosis of Neuronal Cells
Authors: Chuenkova Marina V. , Pereira Miercio A.
Published: July 2013

Despite the neuronal degeneration in the chronic stage of Chagas' disease, neuron counts actually increase in the preceding, asymptomatic stage, in contrast to the age-related decrease in neuron counts in age-matched normal individuals. Relevant to this observation, we found that the trans-sialidase (TS) of Trypanosoma cruzi, the etiologic agent of Chagas' disease, induces neurite outgrowth and rescues PC12 cells from apoptotic death caused by growth factor deprivation. These properties, novel for a parasite protein, were independent of catalytic activity and were mapped to the C terminus of the catalytic domain of TS. TS activated protein kinase Akt in a phosphoinositide-3 kinase-inhibitable manner, suggesting a molecular mechanism for the TS-induced neuroprotection. TS also triggered bcl-2 gene expression in growth factor-deprived cells, an effect consistent with TS protecting against apoptosis. Ciliary neurotrophic factor and leukemia inhibitory factor, two cytokines critical to the repair of injured motor neurons, specifically potentiated the TS action. The results suggest that TS acts in synergy with host ciliary neurotrophic factor or leukemia inhibitory factor to promote neuronal survival in T. cruzi-infected individuals.
Gaa1p and Gpi8p Are Components of a Glycosylphosphatidylinositol (GPI) Transamidase That Mediates Attachment of GPI to Proteins
Authors: Ohishi Kazuhito , Inoue Norimitsu , Maeda Yusuke , Takeda Junji , Riezman Howard , Kinoshita Taroh
Published: July 2013

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted aGAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.
Ca2+Entry through Store-operated Channels in Mouse Sperm Is Initiated by Egg ZP3 and Drives the Acrosome Reaction
Authors: O'Toole Christine M.B. , Arnoult Christophe , Darszon Alberto , Steinhardt Richard A. , Florman Harvey M.
Published: July 2013

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca2+concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca2+concentration increase is due to the persistent activation of a Ca2+influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca2+store depletion–activated Ca2+entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca2+permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca2+influx through a store depletion–operated pathway and that this drives the exocytotic acrosome reaction.
High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes
Authors: Kenworthy Anne K. , Petranova Nadezda , Edidin Michael
Published: July 2013

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.
Identification and Characterization ofSaccharomyces cerevisiaeCdc6 DNA-binding Properties
Authors: Feng Luo , Wang Bin , Driscoll Barbara , Jong Ambrose
Published: July 2013

Recent studies have shown that Cdc6 is an essential regulator in the formation of DNA replication complexes. However, the biochemical nature of the Cdc6 molecule is still largely unknown. In this report, we present evidence that the Saccharomyces cerevisiaeCdc6 protein is a double-stranded DNA-binding protein. First, we have demonstrated that the purified yeast Cdc6 can bind to double-stranded DNA (dissociation constant ∼ 1 × 10−7M), not to single-stranded DNA, and that the Cdc6 molecule is a homodimer in its native form. Second, we show that GST-Cdc6 fusion proteins expressed in Escherichia coli bind DNA in an electrophoretic mobility shift assay. Cdc6 antibodies and GST antibodies, but not preimmune serum, induce supershifts of GST-Cdc6 and DNA complexes in these assays, which also showed that GST-Cdc6 binds to various DNA probes without apparent sequence specificity. Third, the minimal requirement for the binding of Cdc6 to DNA has been mapped within its N-terminal 47-amino acid sequence (the NP6 region). This minimal binding domain shows identical DNA-binding properties to those possessed by full-length Cdc6. Fourth, the GST-NP6 protein competes for DNA binding with distamycin A, an antibiotic that chelates DNA within the minor groove of the A+T-rich region. Finally, site-direct mutagenesis studies revealed that the29KRKK region of Cdc6 is essential for Cdc6 DNA-binding activity. To further elucidate the function of Cdc6 DNA binding in vivo, we demonstrated that a binding mutant of Cdc6 fails to complement either cdc6-1 temperature-sensitive mutant cells orΔcdc6 null mutant cells at the nonpermissive temperature. The mutant gene also conferred growth impairments and increased the plasmid loss in its host, indicative of defects in DNA synthesis. Because the mutant defective in DNA binding also fails to stimulate Abf1 ARS1 DNA-binding activity, our results suggest that Cdc6 DNA-binding activity may play a pivotal role in the initiation of DNA replication.
HRDGene Dependence of Endoplasmic Reticulum-associated Degradation
Authors: Wilhovsky Sharon , Gardner Richard , Hampton Randolph
Published: July 2013

Work from several laboratories has indicated that many different proteins are subject to endoplasmic reticulum (ER) degradation by a common ER-associated machinery. This machinery includes ER membrane proteins Hrd1p/Der3p and Hrd3p and the ER-associated ubiquitin-conjugating enzymes Ubc7p and Ubc6p. The wide variety of substrates for this degradation pathway has led to the reasonable hypothesis that the HRD (Hmg CoA reductase degradation) gene-encoded proteins are generally involved in ER protein degradation in eukaryotes. We have tested this model by directly comparing the HRD dependency of the ER-associated degradation for various ER membrane proteins. Our data indicated that the role of HRD genes in protein degradation, even in this highly defined subset of proteins, can vary from absolute dependence to complete independence. Thus, ER-associated degradation can occur by mechanisms that do not involve Hrd1p or Hrd3p, despite their apparently broad envelope of substrates. These data favor models in which the HRD gene-encoded proteins function as specificity factors, such as ubiquitin ligases, rather than as factors involved in common aspects of ER degradation.
Activation of Cdc42, Rac, PAK, and Rho-Kinase in Response to Hepatocyte Growth Factor Differentially Regulates Epithelial Cell Colony Spreading and Dissociation
Authors: Royal Isabelle , Lamarche-Vane Nathalie , Lamorte Louie , Kaibuchi Kozo , Park Morag
Published: July 2013

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial–mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial–mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.
Mammalian Spindle Orientation and Position Respond to Changes in Cell Shape in a Dynein-dependent Fashion
Authors: O'Connell Christopher B. , Wang Yu-li
Published: July 2013

In animal cells, positioning of the mitotic spindle is crucial for defining the plane of cytokinesis and the size ratio of daughter cells. We have characterized this phenomenon in a rat epithelial cell line using microscopy, micromanipulation, and microinjection. Unmanipulated cells position the mitotic spindle near their geometric center, with the spindle axis lying roughly parallel to the long axis of the cell. Spindles that were initially misoriented underwent directed rotation and caused a delay in anaphase onset. To gain further insight into this process, we gently deformed cells with a blunted glass needle to change the spatial relationship between the cortex and spindle. This manipulation induced spindle movement or rotation in metaphase and/or anaphase, until the spindle reached a proper position relative to the deformed shape. Spindle positioning was inhibited by either treatment with low doses of nocodazole or microinjection of antibodies against dynein, apparently due to the disruption of the organization of dynein and/or astral microtubules. Our results suggest that mitotic cells continuously monitor and maintain the position of the spindle relative to the cortex. This process is likely driven by interactions among astral microtubules, the motor protein dynein, and the cell cortex and may constitute part of a mitotic checkpoint mechanism.
Sorting to Synaptic-like Microvesicles from Early and Late Endosomes Requires Overlapping but Not Identical Targeting Signals
Authors: Blagoveshchenskaya Anastasiya D. , Cutler Daniel F.
Published: July 2013

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.
Involvement of a Small GTP-binding Protein (G Protein) Regulator, Small G Protein GDP Dissociation Stimulator, in Antiapoptotic Cell Survival Signaling
Authors: Takakura Ayumi , Miyoshi Jun , Ishizaki Hiroyoshi , Tanaka Miki , Togawa Atsushi , Nishizawa Yasuko , Yoshida Hisahiro , Nishikawa Shin-ichi , Takai Yoshimi
Published: July 2013

Small GTP-binding protein GDP dissociation stimulator (Smg GDS) regulates GDP/GTP exchange reaction of Ki-Ras and the Rho and Rap1 family members and inhibits their binding to membranes. In fibroblasts, Smg GDS shows mitogenic and transforming activities in cooperation with Ki-Ras. However, the physiological function of Smg GDS remains unknown. Here we show that mice lacking Smg GDS died of heart failure shortly after birth, not resulting from developmental heart defects but from enhanced apoptosis of cardiomyocytes triggered by cardiovascular overload. Furthermore, neonatal thymocytes and developing neuronal cells underwent apoptotic cell death. Smg GDS−/− thymocytes were susceptible to apoptotic inducers, such as etoposide and UV irradiation. Smg GDS−/− thymocytes were protected from etoposide-induced cell death by ex vivo transduction of the Smg GDS cDNA. These phenotypes partly coincide with those observed in Ki-Ras-deficient mice, suggesting that Smg GDS is involved in antiapoptotic cell survival signaling through Ki-Ras.
Branched Chain Amino Acids Induce Apoptosis in Neural Cells without Mitochondrial Membrane Depolarization or CytochromecRelease: Implications for Neurological Impairment Associated with Maple Syrup Urine Disease
Authors: Jouvet Philippe , Rustin Pierre , Taylor Deanna L. , Pocock Jennifer M. , Felderhoff-Mueser Ursula , Mazarakis Nicholas D. , Sarraf Catherine , Joashi Umesh , Kozma Mary , Greenwood Kirsty , Edwards A. David , Mehmet Huseyin
Published: July 2013

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain α-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular α-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, α-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their α-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.
Cytoplasmic Dynein-mediated Assembly of Pericentrin and γ Tubulin onto Centrosomes
Authors: Young Aaron , Dictenberg Jason B. , Purohit Aruna , Tuft Richard , Doxsey Stephen J.
Published: July 2013

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and γ tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and γ tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and γ tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where γ tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and γ tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.
Histone Deacetylase Inhibitors Trigger a G2 Checkpoint in Normal Cells That Is Defective in Tumor Cells
Authors: Qiu Ling , Burgess Andrew , Fairlie David P. , Leonard Helen , Parsons Peter G. , Gabrielli Brian G.
Published: July 2013

Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity. An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers. Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines. Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death. This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states. Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells.
The Peroxin Pex3p Initiates Membrane Assembly in Peroxisome Biogenesis
Authors: Ghaedi Kamran , Tamura Shigehiko , Okumoto Kanji , Matsuzono Yuji , Fujiki Yukio
Published: July 2013

Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed ∼25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA ratPEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly138and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated.
Mitochondria-to-Nuclear Signaling Is Regulated by the Subcellular Localization of the Transcription Factors Rtg1p and Rtg3p
Authors: Sekito Takayuki , Thornton Janet , Butow Ronald A.
Published: July 2013

Cells modulate the expression of nuclear genes in response to changes in the functional state of mitochondria, an interorganelle communication pathway called retrograde regulation. In yeast, expression of the CIT2 gene shows a typical retrograde response in that its expression is dramatically increased in cells with dysfunctional mitochondria, such as in ρopetites. Three genes control this signaling pathway: RTG1 andRTG3, which encode basic helix-loop-helix leucine zipper transcription factors that bind as heterodimer to theCIT2 upstream activation site, and RTG2, which encodes a protein of unknown function. We show that in respiratory-competent (ρ+) cells in whichCIT2 expression is low, Rtg1p and Rtg3p exist as a complex largely in the cytoplasm, and in ρopetites in which CIT2 expression is high, they exist as a complex predominantly localized in the nucleus. Cytoplasmic Rtg3p is multiply phosphorylated and becomes partially dephosphorylated when localized in the nucleus. Rtg2p, which is cytoplasmic in both ρ+and ρocells, is required for the dephosphorylation and nuclear localization of Rtg3p. Interaction of Rtg3p with Rtg1p is required to retain Rtg3p in the cytoplasm of ρ+cells; in the absence of such interaction, nuclear localization and dephosphorylation of Rtg3p is independent of Rtg2p. Our data show that Rtg1p acts as both a positive and negative regulator of the retrograde response and that Rtg2p acts to transduce mitochondrial signals affecting the phosphorylation state and subcellular localization of Rtg3p.
Density-dependent Growth Inhibition of Fibroblasts Ectopically Expressing p27kip1
Authors: Zhang Xiaohong , Wharton Walker , Donovan Marcia , Coppola Domenico , Croxton Rhonda , Cress W. Douglas , Pledger W. J.
Published: July 2013

The cyclin/cyclin-dependent kinase (cdk) inhibitor p27kip1 is thought to be responsible for the onset and maintenance of the quiescent state. It is possible, however, that cells respond differently to p27kip1 in different conditions, and using a BALB/c-3T3 cell line (termed p27-47) that inducibly expresses high levels of this protein, we show that the effect of p27kip1 on cell cycle traverse is determined by cell density. We found that ectopic expression of p27kip1blocked the proliferation of p27-47 cells at high density but had little effect on the growth of cells at low density whether exponentially cycling or stimulated from quiescence. Regardless of cell density, the activities of cdk4 and cdk2 were markedly repressed by p27kip1 expression, as was the cdk4-dependent dissociation of E2F4/p130 complexes. Infection of cells with SV40, a DNA tumor virus known to abrogate formation of p130- and Rb-containing complexes, allowed dense cultures to proliferate in the presence of supraphysiological amounts of p27kip1 but did not stimulate cell cycle traverse when cultures were cotreated with the potent cdk2 inhibitor roscovitine. Our data suggest that residual levels of cyclin/cdk activity persist in p27kip1-expressing p27-47 cells and are sufficient for the growth of low-density cells and of high-density cells infected with SV40, and that effective disruption of p130 and/or Rb complexes is obligatory for the proliferation of high-density cultures.
Reversible Block of the Calcium Release Channel/Ryanodine Receptor by Protamine, a Heparin Antidote
Authors: Koulen Peter , Ehrlich Barbara E.
Published: July 2013

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C2C12mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.
Maintenance of Human Rearranged Mitochondrial DNAs in Long-Term Cultured Transmitochondrial Cell Lines
Authors: Tang Yingying , Manfredi Giovanni , Hirano Michio , Schon Eric A.
Published: July 2013

Large-scale rearrangements of mitochondrial DNA (mtDNA; i.e., partial duplications [dup-mtDNAs] and deletions [Δ-mtDNAs]) coexist in tissues in a subset of patients with sporadic mitochondrial disorders. In order to study the dynamic relationship among rearranged and wild-type mtDNA (wt-mtDNA) species, we created transmitochondrial cell lines harboring various proportions of wt-, Δ-, and dup-mtDNAs from two patients. After prolonged culture in nonselective media, cells that contained initially 100% dup-mtDNAs became heteroplasmic, containing both wild-type and rearranged mtDNAs, likely generated via intramolecular recombination events. However, in cells that contained initially a mixture of both wt- and Δ-mtDNAs, we did not observe any dup-mtDNAs or other new forms of rearranged mtDNAs, perhaps because the two species were physically separated and were therefore unable to recombine. The ratio of wt-mtDNA to Δ-mtDNAs remained stable in all cells examined, suggesting that there was no replicative advantage for the smaller deleted molecules. Finally, in cells containing a mixture of monomeric and dimeric forms of a specific Δ-mtDNA, we found that the mtDNA population shifted towards homoplasmic dimers, suggesting that there may be circumstances under which the cells favor molecules with multiple replication origins, independent of the size of the molecule.
Reversible Merger of Membranes at the Early Stage of Influenza Hemagglutinin-mediated Fusion
Authors: Leikina Eugenia , Chernomordik Leonid V.
Published: July 2013

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10–20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37°C distinguish the newly identified fusion intermediate from the intermediate arrested at 4°C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.
Regulation of Membrane Type-1 Matrix Metalloproteinase Activation by Proprotein Convertases
Authors: Yana Ikuo , Weiss Stephen J.
Published: July 2013

Membrane type-1 matrix metalloproteinase (MT1-MMP) is the prototypical member of a subgroup of membrane-anchored proteinases that belong to the matrix metalloproteinase family. Although synthesized as a zymogen, MT1-MMP plays an essential role in extracellular matrix remodeling after an undefined process that unmasks its catalytic domain. We now report the existence of a proprotein convertase–MT1-MMP axis that regulates the processing and functional activity of the metalloproteinase. Two sets of basic motifs in the propeptide region of MT1-MMP are identified that potentially can be recognized by the proprotein convertase family of subtilisin-like proteases. Processing of proMT1-MMP as well as the expression of its proteolytic activity were blocked by mutating these recognition motifs or by inhibiting the proprotein convertases furin and PC6 with the serpin-based inhibitor α1antitrypsin Portland. Furthermore, both furin-dependent and furin-independent MT1-MMP processing pathways are identified that require tethering of the metalloproteinase to the cell surface. These findings demonstrate the existence of a proprotein convertase–MT1-MMP axis that can regulate extracellular matrix remodeling.
Transient Nucleolar Localization Of U6 Small Nuclear RNA InXenopus LaevisOocytes
Authors: Lange Thilo Sascha , Gerbi Susan A.
Published: July 2013

Recent studies on the 2′-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.