Maternally Expressed γTub37CD inDrosophilaIs Differentially Required for Female Meiosis and Embryonic Mitosis

AbstractWe report functional analysis of γTub37CD, a maternally synthesized γ-tubulin that is highly expressed during oogenesis and utilized at centrosomes in precellular embryos. TwoγTub37CDmutants contained missense mutations that altered residues conserved in all γ-tubulins and α- and/or β-tubulins. A thirdγTub37CDmissense mutant identified a conserved motif unique to γ-tubulins. A fourthγTub37CDmutant contained a nonsense mutation and the corresponding premature stop codon generated a protein null allele. Immunofluorescence analysis of laid eggs and activated oocytes derived from the mutants revealed microtubules and meiotic spindles that were close to normal even in the absence of γTub37CD. Eggs lacking the maternal γ-tubulin were arrested in meiosis, indicative of a deficiency in activation. Analysis of meiosis within vitroactivation techniques showed that the cortical microtubule cytoskeleton of mature wild-type eggs was reorganized upon activation and expressed as transient assembly of cortical asters, and this cortical reorganization was altered inγTub37CDmutants. In precellular embryos of partial loss of function mutants, spindles were frequently abnormal and cell cycle progression was inhibited. Thus, γTub37CD functions differentially in female meiosis and in the early embryo; while involved in oocyte activation, it is apparently not required or plays a subtle role in formation of the female meiotic spindle which is acentriolar, but is essential for assembly of a discrete bipolar mitotic spindle which is directed by centrosomes organized about centrioles

Multiplexed spectral imaging of 120 different fluorescent labels

This article is distributed under the terms of the Creative Commons public domain dedication. The definitive version was published in PLoS One 11 (2016): e0158495, doi:10.1371/journal.pone.0158495.The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image.This work was supported by Grant 2007-3- 13 from the Alfred P. Sloan Foundation (to GGB), National Institutes of Health Grant 1RC1-DE020630 from the National Institute of Dental and Craniofacial Research (NIDCR) (to GGB) and by National Institutes of Health Fellowship 1F31-DE019576 from NIDCR (to AMV)

Detyrosination of alpha tubulin does not stabilize microtubules in vivo.

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization

Individuality, stability, and variability of the plaque microbiome

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 564, doi:10.3389/fmicb.2016.00564.Dental plaque is a bacterial biofilm composed of a characteristic set of organisms. Relatively little information from cultivation-independent, high-throughput analyses has been published on the temporal dynamics of the dental plaque microbiome. We used Minimum Entropy Decomposition, an information theory-based approach similar to oligotyping that provides single-nucleotide resolution, to analyze a previously published time series data set and investigate the dynamics of the plaque microbiome at various analytic and taxonomic levels. At both the genus and 97% Operational Taxonomic Unit (OTU) levels of resolution, the range of variation within each individual overlapped that of other individuals in the data set. When analyzed at the oligotype level, however, the overlap largely disappeared, showing that single-nucleotide resolution enables differentiation of individuals from one another without ambiguity. The overwhelming majority of the plaque community in all samples was made up of bacteria from a moderate number of plaque-typical genera, indicating that the overall community framework is shared among individuals. Each of these genera fluctuated in abundance around a stable mean that varied between individuals, with some genera having higher inter-individual variability than others. Thus, at the genus level, differences between individuals lay not in the identity of the major genera but in consistently differing proportions of these genera from mouth to mouth. However, at the oligotype level, we detected oligotype “fingerprints,” a highly individual-specific set of persistently abundant oligotypes fluctuating around a stable mean over time. For example, within the genus Corynebacterium, more than a dozen oligotypes were detectable in each individual, of which a different subset reached high abundance in any given person. This pattern suggests that each mouth contains a subtly different community of organisms. We also compared the Chinese plaque community characterized here to previously characterized Western plaque communities, as represented by analyses of data emerging from the Human Microbiome Project, and found no major differences between Chinese and Western supragingival plaque. In conclusion, we found the plaque microbiome to be highly individualized at the oligotype level and characterized by stability of community membership, with variability in the relative abundance of community members between individuals and over time.Our work was supported by National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research Grant DE022586 (to GGB). Additional support was provided by Harvard University's Department of Organismic and Evolutionary Biology graduate program (to DRU)