Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae. (33/8843)

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.  (+info)

Effect of phosducin on opioid receptor function. (34/8843)

Phosducin (Phd) regulates the function of G proteins by its ability to tightly bind Gbetagamma subunits. Because the internalization of opioid receptors as well as the activity of adenylyl cyclase (AC) activity depends on G proteins, we tested Phd on these parameters. NG 108-15 hybrid cells stably expressing the phosphoprotein were challenged with [D-penicillamine2,D-penicillamine5]enkephalin to inhibit cAMP generation, demonstrating an increased efficacy of the opioid on AC. Studying the binding of [35S]guanosine-5'-O-(gamma-thio)-triphosphate to membranes from Phd overexpressing cells, we found that [D-penicillamine2, D-penicillamine5 ]enkephalin failed, in the presence of Phd (0.1 nM), to elevate incorporation of the nucleotide. Phd also strongly inhibited opioid-stimulated GTPase activity. NG 108-15 cells were also employed to investigate the effect of Phd on opioid receptor internalization. Control cells and cells overexpressing Phd were transiently transfected to express mu-opioid receptors fused to green fluorescence protein. In controls and in Phd overexpressing cells confocal microscopy identified fluorescence associated with the membrane. Time-lapse series microscopy of living control cells challenged with etorphine (1 microM) revealed receptor internalization within 30 min. In contrast, Phd overexpressing cells largely failed to respond to the opioid. Thus, in Phd overexpressing cells, opioids exhibit an increased efficacy despite the inhibitory action of the phosphoprotein on opioid-stimulated incorporation of [35S]guanosine-5'-O-(gamma-thio)-triphosphate. We suggest that inhibition of GTPase stabilizes the opioid-induced G protein Gi-GTP complex, which is believed to enhance AC inhibition. Finally, scavenging of Gbetagamma by Phd attenuates internalization of opioid receptors, which may contribute to the efficacy of opioids.  (+info)

Dynamic redistribution of STAT1 protein in IFN signaling visualized by GFP fusion proteins. (35/8843)

STAT proteins (signal transducers and activators of transcription) are a family of transcription factors which are used by many cytokines and cell growth factors for initiating gene expression. They are activated by tyrosine phosphorylation through the cytoplasmic domain of stimulated receptors. Upon phosphorylation STAT proteins dimerize, translocate to the nucleus and activate transcription by binding to specific recognition sites. Different cytokines activate different subsets of STATs and other signaling proteins. We have made use of green fluoresencent protein (GFP) fusion proteins to visualize the subcellular localization and trafficking of STAT1, STAT2 and p48 during interferon (IFN) stimulation and have analysed in detail STAT1-GFP trafficking in living cells. Analysis of GFP fusion proteins allowed the determination of time kinetics of subcellular trafficking in individual living cells. STAT1-GFP is indistinguishable from its wild-type protein displaying strong activity as transcriptional activator as well as the same time kinetics of transport to the nucleus and retreat to the cytoplasm. After prolonged exposure to IFN, STAT1-GFP is no longer retained in the nucleus and relocation to the cytoplasm is observed. Restimulation with the same type of IFN does not lead to repeated nuclear translocation of STAT1-GFP. STAT1 is not subject of inhibition, as restimulation with another type of IFN allows immediate reuse of previously activated STAT1-GFP. However, restimulation with the same type of IFN can be achieved when the primary stimulus is removed after a short induction period. This method of visualizing signal transduction reveals a considerable inhomogeneity with respect to the extent of STAT1-GFP shuttling within a clonal cell population, indicating that competence for full-blasted IFN response is restricted to a cellular subpopulation whereas other cells respond incompletely, retarded or not at all.  (+info)

Imaging protein kinase Calpha activation in cells. (36/8843)

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.  (+info)

Identification of peroxisomal acyl-CoA thioesterases in yeast and humans. (37/8843)

A computer-based screen of the Saccharomyces cerevisiae genome identified YJR019C as a candidate oleate-induced gene. YJR019C mRNA levels were increased significantly during growth on fatty acids, suggesting that it may play a role in fatty acid metabolism. The YJR019C product is highly similar to tesB, a bacterial acyl-CoA thioesterase, and carries a tripeptide sequence, alanine-lysine-phenylalanineCOOH, that closely resembles the consensus sequence for type-1 peroxisomal targeting signals. YJR019C directed green fluorescence protein to peroxisomes, and biochemical studies revealed that YJR019C is an abundant component of purified yeast peroxisomes. Disruption of the YJR019C gene caused a significant decrease in total cellular thioesterase activity, and recombinant YJR019C was found to exhibit intrinsic acyl-CoA thioesterase activity of 6 units/mg. YJR019C also shared significant sequence similarity with hTE, a human thioesterase that was previously identified because of its interaction with human immunodeficiency virus-Nef in the yeast two-hybrid assay. We report here that hTE is also a peroxisomal protein, demonstrating that thioesterase activity is a conserved feature of peroxisomes. We propose that YJR019C and hTE be renamed as yeast and human PTE1 to reflect the fact that they encode peroxisomal thioesterases. The physical segregation of yeast and human PTE1 from the cytosolic fatty acid synthase suggests that these enzymes are unlikely to play a role in formation of fatty acids. Instead, the observation that PTE1 contributes to growth on fatty acids implicates this thioesterase in fatty acid oxidation.  (+info)

Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin. (38/8843)

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.  (+info)

Distinct regions specify the nuclear membrane targeting of emerin, the responsible protein for Emery-Dreifuss muscular dystrophy. (39/8843)

Emery-Dreifuss muscular dystrophy is a neuromuscular disorder that has three characteristics: (a) early contracture of the elbows, Achilles tendons and postcervical muscles; (b) slowly progressive wasting and weakness of skeletal muscle; and (c) cardiomyopathy with severe conduction block. The responsible gene for the X-linked recessive form of this disease encodes an inner nuclear membrane protein named emerin. Although emerin is absent in tissues from patients with this disorder, it remains obscure why the loss of this widely expressed protein affects selectively skeletal muscle, heart and joints. As the first step to address this question, we examined the molecular regions of emerin that are essential for nuclear membrane targeting and stability of the protein. We found that the C-terminal hydrophobic region was necessary, but not sufficient, for nuclear membrane anchoring and stability of the protein. In the absence of this transmembrane domain, the upstream nucleoplasmic domain showed no firm association with the nuclear rim, but showed the tendency to accumulate at the nucleolus-like structures. Furthermore, proper targeting of emerin to the nuclear membrane required the latter half of the nucleoplasmic domain. These characteristics are distinct from those of lamina-associated polypeptide 2. Our findings indicate that emerin has distinct interactions with the inner nuclear membrane components that may be required for the stability and function of rigorously moving nuclei in tissues such as skeletal muscle, heart and joints.  (+info)

Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion protein. (40/8843)

Directional migration of border cells in the Drosophila egg chambers is a developmentally regulated event that requires dynamic cellular functions. In this study, the electron microscopic observation of migrating border cells revealed loose actin bundles in forepart lamellipodia and numerous microvilli extending from nurse cells and providing multiple adhesive contacts with border cells. To analyze the dynamics of actin in migrating border cells in vivo, we constructed a green fluorescent protein-actin fusion protein and induced its expression in Drosophila using the GAL4/UAS system. The green fluorescent protein-actin was incorporated into the actin bundles and it enabled visualization of the rapid cytoskeletal changes in border cell lamellipodia. During the growth of the lamellipodia, the actin bundles that increased in number and size radiated from the bundle-organizing center. Quantification of the fluorescence intensity showed that an accumulation of bundle-associated and spotted green fluorescent protein-actin signals took place during their centripetal movement. Our results favored a treadmilling model for actin behavior in border cell lamellipodia.  (+info)