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.

Mammalian Transcription Factor ATF6 Is Synthesized as a Transmembrane Protein and Activated by Proteolysis in Response to Endoplasmic Reticulum Stress

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

Sequential Actions of Phospholipase D and Phosphatidic Acid Phosphohydrolase 2b Generate Diglyceride in Mammalian Cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1–enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.

The Dynamin-like Protein DLP1 Is Essential for Normal Distribution and Morphology of the Endoplasmic Reticulum and Mitochondria in Mammalian Cells

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Network of tangential pathways for neuronal migration in adult mammalian brain

Cells in the brains of adult mammals continue to proliferate in the subventricular zone (SVZ) throughout the lateral wall of the lateral ventricle. Here we show, using whole mount dissections of this wall from adult mice, that the SVZ is organized as an extensive network of chains of neuronal precursors. These chains are immunopositive to the polysialylated form of NCAM, a molecule present at sites of plasticity, and TuJ1, an early neuronal marker. The majority of the chains are oriented along the rostrocaudal axis and many join the rostral migratory stream that terminates in the olfactory bulb. Using focal microinjections of DiI and transplantation of SVZ cells carrying a neuron-specific reporter gene, we demonstrate that cells originating at different rostrocaudal levels of the SVZ migrate rostrally and reach the olfactory bulb where they differentiate into neurons. Our results reveal an extensive network of pathways for the tangential chain migration of neuronal precursors throughout the lateral wall of the lateral ventricle in the adult mammalian brain.

The number of unidentified amacrine cells in the mammalian retina

The three largest known populations of amacrine cells in the rabbit retina were stained with fluorescent probes in whole mounts and counted at a series of retinal eccentricities. The retinas were counterstained using a fluorescent DNA-binding molecule and the total number of nuclei in the inner nuclear layer were counted in confocal sections. From the total number of inner nuclear layer cells and the known fraction of them occupied by amacrine cells, the fraction of amacrine cells made up by the stained populations could be calculated. Starburst cells made up 3%, indoleamine-accumulating cells made up 4%, and AII cells made up 11% of all amacrine cells. By referring four smaller populations of amacrine cells to the number of indoleamine-accumulating cells, they were estimated to make up 4% of all amacrine cells. Thus, 78% of all amacrine cells in the rabbit’s retina are known only from isolated examples, if at all. This proportion is similar in the retinas of the mouse, cat, and monkey. It is likely that a substantial fraction of the local circuit neurons present in other regions of the central nervous system are also invisible as populations to current techniques.

Evolutionary conservation of apoptosis mechanisms: Lepidopteran and baculoviral inhibitor of apoptosis proteins are inhibitors of mammalian caspase-9

We cloned a new inhibitor of apoptosis protein (IAP) homolog, SfIAP, from Spodoptera frugiperda Sf-21 cells, a host of insect baculoviruses. SfIAP contains two baculovirus IAP repeat domains followed by a RING domain. SfIAP has striking amino acid sequence similarity with baculoviral IAPs, CpIAP and OpIAP, suggesting that baculoviral IAPs may be host-derived genes. SfIAP and baculoviral CpIAP inhibit Bax but not Fas-induced apoptosis in human cells. Their apoptosis-suppressing activity in mammalian cells requires both baculovirus IAP repeat and RING domains. Further biochemical data suggest that SfIAP and CpIAP are specific inhibitors of mammalian caspase-9, the pinnacle caspase in the mitochondria/cytochrome c pathway for apoptosis, but are not inhibitors of downstream caspase-3 and caspase-7. Thus the mechanisms by which insect and baculoviral IAPs suppress apoptosis may involve inhibition of an insect caspase-9 homologue. Peptides representing the IAP-binding domain of the Drosophila cell death protein Grim abrogated human caspase suppression by SfIAP and CpIAP, implying evolutionary conservation of the functions of IAPs and their inhibitors.

Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage

A sensitive and rapid in situ method was developed to visualize sites of single-stranded (ss) DNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form. After treatment of cells with DNA-damaging agents or γ irradiation, ssDNA molecules form nuclear foci in a dose-dependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombinational DNA repair.

Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+ channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+ channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+ channel gene. Paradoxically, we find that addition of the extra K+ channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+ channel gene dosage leads to dysregulation of normal K+ channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.