Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.

Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the α-ketoglutarate dehydrogenase complex (αKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of αKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of αKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.

Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis

Accumulated evidence attributes noncatalytic morphogenic activitie(s) to acetylcholinesterase (AChE). Despite sequence homologies, functional overlaps between AChE and catalytically inactive AChE-like cell surface adhesion proteins have been demonstrated only for the Drosophila protein neurotactin. Furthermore, no mechanism had been proposed to enable signal transduction by AChE, an extracellular enzyme. Here, we report impaired neurite outgrowth and loss of neurexin Iα mRNA under antisense suppression of AChE in PC12 cells (AS-ACHE cells). Neurite growth was partially rescued by addition of recombinant AChE to the solid substrate or by transfection with various catalytically active and inactive AChE variants. Moreover, overexpression of the homologous neurexin I ligand, neuroligin-1, restored both neurite extension and expression of neurexin Iα. Differential PCR display revealed expression of a novel gene, nitzin, in AS-ACHE cells. Nitzin displays 42% homology to the band 4.1 protein superfamily capable of linking integral membrane proteins to the cytoskeleton. Nitzin mRNA is high throughout the developing nervous system, is partially colocalized with AChE, and increases in rescued AS-ACHE cells. Our findings demonstrate redundant neurite growth-promoting activities for AChE and neuroligin and implicate interactions of AChE-like proteins and neurexins as potential mediators of cytoarchitectural changes supporting neuritogenesis.

Subunit structure of the mammalian exocyst complex

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis.

Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response

Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpT-treated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6–4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

In vitro-generated neural precursors participate in mammalian brain development

During embryogenesis, pluripotent stem cells segregate into daughter lineages of progressively restricted developmental potential. In vitro, this process has been mimicked by the controlled differentiation of embryonic stem cells into neural precursors. To explore the developmental potential of these cell-culture-derived precursors in vivo, we have implanted them into the ventricles of embryonic rats. The transplanted cells formed intraventricular neuroepithelial structures and migrated in large numbers into the brain tissue. Embryonic-stem-cell-derived neurons, astrocytes, and oligodendrocytes incorporated into telencephalic, diencephalic, and mesencephalic regions and assumed phenotypes indistinguishable from neighboring host cells. These observations indicate that entirely in vitro-generated neural precursors are able to respond to environmental signals guiding cell migration and differentiation and have the potential to reconstitute neuronal and glial lineages in the central nervous system.

OBA/Ku86: DNA Binding Specificity and Involvement in Mammalian DNA Replication

Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4–OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of ∼92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication.

Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian CellsV⃞

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1− yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1− yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA. As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA. For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant. We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts. We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified.

Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network.

Differential Distribution of Dynamin Isoforms in Mammalian Cells

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.