Biology subjects

Howard, J.

Publications and source records attributed to Howard, J..

4 recordsLinked to original sources

bioRxiv2018

The dynamic and structural properties of axonemal tubulins support the high length stability of cilia

Cilia and flagella, which play essential roles in cell motility, sensing and development, contain at their core a microtubule-based structure called axoneme. The axoneme, which contains nine doublet and two central microtubules, is highly stable and its length is precisely-controlled. We have asked whether the axonemal tubulins contribute to this length stability. Towards this end, we used a novel procedure to differentially extract the tubulins from the different axoneme components, and characterized their dynamic properties and post-translational modifications (PTMs). We found that their dynamic properties are consistent with the greater stability of axonemes. Despite the differences in PTMs between the axonemal components, we found no significant differences in their dynamic properties. Unexpectedly, the reconstituted ciliary microtubules exhibit curved protofilaments at their growing tip, that may correspond to the fluctuating GTP-cap hypothesized to exist. Thus, our study provides new insights into the growth, stability and the role of PTMs of ciliary tubulin.\n\nAbbreviations

biochemistry↗

bioRxiv2018

Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

biophysics↗

bioRxiv2017

De Novo PacBio long-read and phased avian genome assemblies correct and add to genes important in neuroscience research

Reference quality genomes are expected to provide a resource for studying gene structure and function. However, often genes of interest are not completely or accurately assembled, leading to unknown errors in analyses or additional cloning efforts for the correct sequences. A promising solution to this problem is long-read sequencing. Here we tested PacBio-based long-read sequencing and diploid assembly for potential improvements to the Sanger-based intermediate-read zebra finch reference and Illumina-based short-read Annas hummingbird reference, two vocal learning avian species widely studied in neuroscience and genomics. With DNA of the same individuals used to generate the reference genomes, we generated diploid assemblies with the FALCON-Unzip assembler, resulting in contigs with no gaps in the megabase range (N50s of 5.4 and 7.7 Mb, respectively), and representing 150-fold and 200-fold improvements over the current zebra finch and hummingbird references, respectively. These long-read assemblies corrected and resolved what we discovered to be misassemblies, including due to erroneous sequences flanking gaps, complex repeat structure errors in the references, base call errors in difficult to sequence regions, and inaccurate resolution of allelic differences between the two haplotypes. We analyzed protein-coding genes widely studied in neuroscience and specialized in vocal learning species, and found numerous assembly and sequence errors in the reference genes that the PacBio-based assemblies resolved completely, validated by single long genomic reads and transcriptome reads. These findings demonstrate, for the first time in non-human vocal learning species, the impact of higher quality, phased and gap-less assemblies for understanding gene structure and function.

genomics↗

bioRxiv2016

mRNA turnover through CDS-targeting is the primary role of miRNA in the green alga Chlamydomonas.

MicroRNAs regulate gene expression as part of the RNA-induced silencing complex, where the sequence identity of the miRNA provides the specificity to the target messenger RNA, and the result is target repression. The mode of repression can be through target cleavage, RNA destabilization and/or decreased translational efficiency. Here, we provide a comprehensive global analysis of the evolutionarily distant unicellular green alga Chlamydomonas reinhardtii to quantify the effects of miRNA on protein synthesis and RNA abundance. We show that, similar to metazoan systems, miRNAs in Chlamydomonas regulate gene-expression primarily by destabilizing mRNAs. However, unlike metazoan miRNA where target site utilization localizes mainly to 3UTRs, in Chlamydomonas utilized target sites lie predominantly within coding regions. These results demonstrate that destabilization of mRNA is the main evolutionarily conserved mode of action for miRNAs, but details of the mechanism diverge between plant and metazoan kingdoms.

molecular biology↗