Regulation of intrinsic polarity establishment by a differentiation-type MAPK pathway in S. cerevisiae.

All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in the yeast Saccharomyces cerevisiae, which is controlled by the Rho GTPase Cdc42p. The prevailing view of bud emergence does not account for regulation by extrinsic cues. Here, we show that the filamentous growth mitogen activated protein kinase (fMAPK) pathway regulates bud emergence under nutrient-limiting conditions. The fMAPK pathway regulated the expression of polarity targets including the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and active during the period of the cell cycle leading up to bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

Mutations in cytochrome c oxidase subunit VIa cause neurodegeneration and motor dysfunction in Drosophila.

Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy(+) gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COX-mediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.

Untranslated regions of FbRbcS1 mRNA mediate bundle sheath cell-specific gene expression in leaves of a C4 plant.

C4 photosynthesis typically requires two specialized leaf cell types, bundle sheath (bs) and mesophyll (mp), which provide the foundation for this highly efficient carbon assimilation pathway. In leaves of Flaveria bidentis, a dicotyledonous C4 plant, ribulose 1,5-bisphosphate carboxylase (rubisco) accumulates only in bs cells surrounding the vascular centers and not in mp cells. This is in contrast to the more common C3 plants, which accumulate rubisco in all photosynthetic cells. Many previous studies have focused on transcriptional control of C4 cell type-specificity; however, post-transcriptional regulation has also been implicated in the bs-specific expression of genes encoding the rubisco subunits. In this current study, a biolistic leaf transformation assay has provided direct evidence that the 5'- and 3'-untranslated regions (UTRs) of F. bidentis FbRbcS1 mRNA (from a nuclear gene encoding the rubisco small subunit), in themselves, confer strong bs cell-specific expression to gfpA reporter gene transcripts when transcribed from a constitutive CaMV promoter. In transformed leaf regions, strong bs cell-specific GFP expression was accompanied by corresponding bs cell-specific accumulation of the constitutively transcribed FbRbcS1 5'-UTR-gfpA-3'-UTR mRNAs. Control constructs lacking any RbcS mRNA sequences were expressed in all leaf cell types. These findings demonstrate that characteristic cell type-specific FbRbcS1 expression patterns in C4 leaves can be established entirely by sequences contained within the transcribed UTRs of FbRbcS1 mRNAs. We conclude that selective transcript stabilization (in bs cells) or degradation (in mp cells) plays a key role in determining bs cell-specific localization of the rubisco enzyme.

Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase.

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase.

Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes.

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.

Nuclear matrix bound fibroblast growth factor receptor is associated with splicing factor rich and transcriptionally active nuclear speckles.

We have used confocal microscopy combined with computer image analysis to evaluate the functional significance of a constitutively expressed form of the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) in the nucleus of rapidly proliferating serum stimulated TE 671 cells, a medullobastoma human cell line. Our results demonstrate a limited number of large sites and numerous smaller sites of FGFR1 in the nuclear interior. The larger sites showed virtually complete colocalization (>90%) with splicing factor rich nuclear speckles while the smaller sites showed very limited overlap (<20%). Similar results were found for several other proliferating cell lines grown in culture. An in situ transcription assay was used to determine colocalization with transcription sites by incorporating 5-bromouridine triphosphate (BrUTP) followed by dual staining for BrUTP and FGFR1. These results combined with those from using an antibody against the large subunit of RNA polymerase II suggest a significant degree of colocalization (26-38%) over both the large and small sites. No colocalization was detected with sites of DNA replication. The spatial arrangements of FGFR1 sites and colocalization with nuclear speckles were maintained following extraction for nuclear matrix. Moreover, immunoblots indicated a significant enrichment of FGFR1 in the nuclear matrix fraction. Our findings suggest an involvement of a nuclear matrix bound FGFR1 in transcriptional and RNA processing events in the cell nucleus. We further propose that nuclear speckles, aside from a role in transcriptional/RNA processing events, may serve as fundamental regulatory factories for the integration of diverse signaling and regulatory factors that impact transcription and cellular regulation.