Louisa Howard

The Formation of Stable Rhodopsin-Arrestin Complexes Induces Apoptosis and Photoreceptor Cell Degeneration

Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. We now demonstrate the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, we show that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. We propose that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders.

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.

The cohesion protein ORD is required for homologue bias during meiotic recombination

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. We conclude that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila.

Functional Characterization of the GATA Transcription Factors GNC and CGA1 Reveals Their Key Role in Chloroplast Development, Growth, and Division in Arabidopsis

Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues.

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

Polarized fluorescence microscopy reveals that septins across diverse species assemble into similar higher-order structures consisting of dynamic, paired filaments.

The filament-forming protein Pil1 assembles linear eisosomes in fission yeast

Eisosomes generate spatial domains in the plasma membrane of yeast cells. The core eisosome protein Pil1 is shown to form filaments in vitro and in cells. Pil1 filaments are stable at the cell cortex, and cytoplasmic Pil1 filament rods appear upon overexpression. This shows a role for self-assembly in organizing cortical domains.

Septin ring size scaling and dynamics require the coiled-coil region of Shs1p

How the size and dynamics of higher-order septin structures is determined is not well understood in any system. In this paper, we show that the coiled-coil domain of the septin Shs1p limits septin ring size and dynamics in the filamentous fungus Ashbya gossypii, providing a link between protein exchange and the scaling of septin assemblies.

Ion migration from interiors to outer surfaces of balsam fir needles during dry, interstorm periods

Abstract When foliage is exposed to rainwater the resulting solutions can be enriched or diluted with respect to certain ionic species. In the case of enrichment, it is difficult to estimate what proportion of ions has been derived from internal tissue pools versus those deposited from the atmosphere. This distinction is critical in interpreting whole-canopy interactions with rainwater, or in estimating dry deposition by leaf rinse methods. This research tested the relative importance of ion migration to surfaces of Abies balsamea (L.) Mill. during dry, interstorm periods. X-ray analysis of needles attached to living trees exposed only to filtered air for 28 days showed that this contribution was relatively small. Most of the excess ions collected in initial rinses or throughfall are derived from dry deposition during interstorm periods.

Protein methylation in full length Chlamydomonas flagella

Post-translational protein modification occurs extensively in eukaryotic flagella. Here we examine protein methylation, a protein modification that has only recently been reported to occur in flagella [Schneider MJ, Ulland M, Sloboda RD.2008. Mol Biol Cell 19(10):4319-4327.]. The cobalamin (vitamin B12) independent form of the enzyme methionine synthase (MetE), which catalyzes the final step in methionine production, is localized to flagella. Here we demonstrate, using immunogold scanning electron microscopy, that MetE is bound to the outer doublets of the flagellum. Methionine can be converted to S-adenosyl methionine, which then serves as the methyl donor for protein methylation reactions. Using antibodies that recognize symmetrically or asymmetrically methylated arginine residues, we identify three highly methylated proteins in intact flagella: two symmetrically methylated proteins of about 30 and 40 kDa, and one asymmetrically methylated protein of about 75 kDa. Several other relatively less methylated proteins could also be detected. Fractionation and immunoblot analysis shows that these proteins are components of the flagellar axoneme. Immunogold thin section electron microscopy indicates that the symmetrically methylated proteins are located in the central region of the axoneme, perhaps as components of the central pair complex and the radial spokes, while the asymmetrically methylated proteins are associated with the outer doublets. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Pulmonary microRNA profiling: implications in upper lobe predominant lung disease

BackgroundNumerous pulmonary diseases manifest with upper lobe predominance including cystic fibrosis, smoking-related chronic obstructive pulmonary disease, and tuberculosis. Zonal hypoxia, characteristic of these pulmonary maladies, and oxygen stress in general is known to exert profound effects on various important aspects of cell biology. Lung macrophages are major participants in the pulmonary innate immune response and regional differences in macrophage responsiveness to hypoxia may contribute in the development of lung disease. MicroRNAs are ubiquitous regulators of human biology and emerging evidence indicates altered microRNA expression modulates respiratory disease processes. The objective of this study is to gain insight into the epigenetic and cellular mechanisms influencing regional differences in lung disease by investigating effect of hypoxia on regional microRNA expression in the lung.All studies were performed using primary alveolar macrophages (n = 10) or bronchoalveolar lavage fluid (n = 16) isolated from human subjects. MicroRNA was assayed via the NanoString nCounter microRNA assay.ResultsDivergent molecular patterns of microRNA expression were observed in alternate lung lobes, specifically noted was disparate expression of miR-93 and miR-4454 in alveolar macrophages along with altered expression of miR-451a and miR-663a in bronchoalveolar lavage fluid. Gene ontology was used to identify potential downstream targets of divergent microRNAs. Targets include cytokines and matrix metalloproteinases, molecules that could have a significant impact on pulmonary inflammation and fibrosis.ConclusionsOur findings show variant regional microRNA expression associated with hypoxia in alveolar macrophages and BAL fluid in the lung—upper vs lower lobe. Future studies should address whether these specific microRNAs may act intracellularly, in a paracrine/endocrine manner to direct the innate immune response or may ultimately be involved in pulmonary host-to-pathogen trans-kingdom cross-talk.