Rachel A. Howard-Till

The lumens of the highly stable microtubules that make up the core of basal bodies, cilia, and flagella are coated with a network of proteins known as MIPs, or microtubule inner proteins. MIPs are hypothesized to enhance the rigidity and stability of these microtubules, but how they assemble and contribute to cilia function is poorly understood. Here we describe a ciliate specific MIP, RIB22, in Tetrahymena thermophila. RIB22 is a calmodulin-like protein found in the A-tubule of doublet and triplet microtubules in cilia and basal bodies. Its localization is dependent on the conserved MIP RIB72. Here we use cryogenic electron tomography (cryoET) to examine RIB22 and its interacting partners in axonemes and basal bodies. RIB22 forms a ternary complex with the C-terminal EF-hand domain of RIB72A and another MIP, FAM166A. Tetrahymena strains lacking RIB22 or the EF-hand domain of RIB72A showed impaired cilia function. CryoET on axonemes from these strains demonstrated an interdependence of the three proteins for stabilization within the structure. Deletion of the RIB72A EF-hand domain resulted in the apparent loss of multiple MIPs in the region. These findings emphasize the intricacy of the MIP network and the importance of understanding MIPs’ functions during cilium assembly and regulation.

Genetic characterization of wild and cultivated plants provides valuable knowledge for conservation and agriculture. DNA sequencing technologies are improving, and costs are dropping. Yet analysis ... METHOD SUMMARY The authors describe a suite of low-cost, do-it-yourself methods for field collection of plant tissues, extraction of genomic DNA suitable for next-generation sequencing and homemade...

Genetic characterization of wild and cultivated plants provides valuable knowledge for conservation and agriculture. DNA sequencing technologies are improving and costs are dropping. Yet, analysis of many species is hindered because they grow in regions that lack infrastructure for advanced molecular biology. We developed and adapted low-cost methods that address these issues. Tissue is collected and stored in silica-gel, avoiding the need for liquid nitrogen and freezers. We have optimized low-cost home-made DNA extraction to increase yields, reduce costs, and produce DNA suitable for next generation sequencing. We also describe how to build a gel documentation system for DNA quantification. As a proof of principle, we use these methods to evaluate wild Berberis darwinii , native to Southern Chile. Method summary: We describe a suite of low-cost do-it-yourself methods for field collection of plant tissues, extraction of genomic DNA suitable for next generation sequencing, and home-made agarose gel documentation suitable for DNA quantification. These methods enable the collection and preparation of samples for genomic analysis in regions with limited infrastructure.

This protocol describes how to build your own agarose gel documentation system to create digital images for quantification of DNA stained with dyes that fluoresce under blue LED light. Free software and inexpensive materials are used that can save tens of thousands of dollars when compared to similar commercial products.

This protocol describes the use of freely available software for the reference-independent analysis of RADseq data produced from a single sample. It is useful to perform a pilot experiment on a single sample for species lacking a reference genome sequence in order to facilitate proper experimental design for a larger-scale experiment. The example data used in this protocol is from a wild Berberis darwinii plant growing in Chile. The protocol produces data that provides an estimation of the number of unique base pairs recovered from a size selection of restriction endonuclease digested genomic DNA, and also an estimation of the frequency of heterozygous polymorphisms in the plant. Evaluation of sequence quality also provides a validation of the methodologies used for tissue collection, DNA extraction, restriction digestion, and size selection (see https://dx.doi.org/10.17504/protocols.io.bdg9i3z6). In addition, the discovered heterozyygous polymorphisms serve as markers to evaluate natural propagation (e.g. self-fertilization versus outcrossing versus clonal propagation) and also for use in tracking the success of human-directed crosses for breeding.

This protocol describes low-cost methods for the collection and desiccation of plant tissues and subsequent extraction of genomic DNA. It contains modifications of previously described methods found at https://link.springer.com/book/10.1007/978-3-319-16259-1 and https://link.springer.com/chapter/10.1007/978-3-319-45021-6_14. We have tested this protocol with leaf tissue from flax, Lupinus luteus, Camelina sativa and Berberis darwinii. DNA is suitable for routine molecular biology assays such as PCR and also for next generation sequencing.

Condensins are highly conserved proteins that are important for chromosome maintenance in nearly all forms of life. While many organisms employ two forms of the condensin complex, the condensin genes in Tetrahymena have expanded even further. Here we report a form of condensin that is specifically active during sexual reproduction. This complex, condensin D, is composed of the core condensin proteins, Smc2 and Smc4, and two unique subunits, the kleisin Cph5 and Cpd2. Cpd2 is also found in somatic nuclei in vegetative cells, but is dispensable for growth and nuclear division. Immunoprecipitation experiments show that condensin D interacts with a putative member of a chromatin remodeling complex during development. Condensin D is required for sexual reproduction and for endo-replication and genome reduction of the progeny's somatic nuclei. Altogether, Tetrahymena possesses at least four forms of condensin to fulfill the needs of maintaining chromosomes in two different nuclei containing the somatic and germline genomes.

In order to understand its diverse functions, we have studied cohesin in the evolutionarily distant ciliate model organism Tetrahymena thermophila. In this binucleate cell, the heritable germline genome is maintained separately from the transcriptionally active somatic genome. In a previous study, we showed that a minimal cohesin complex in Tetrahymena consisted of homologs of Smc1, Smc3, and Rec8, which are present only in the germline nucleus, where they are needed for normal chromosome segregation as well as meiotic DNA repair. In this study, we confirm that a putative homolog of Scc3 is a member of this complex. In the absence of Scc3, Smc1 and Rec8 fail to localize to germline nuclei, Rec8 is hypo-phosphorylated, and cells show phenotypes similar to depletion of Smc1 and Rec8. We also identify a homolog of Scc2, which in other organisms is part of a heterodimeric complex (Scc2/Scc4) that helps load cohesin onto chromatin. In Tetrahymena, Scc2 interacts with Rec8 and Scc3, and its absence causes defects in mitotic and meiotic divisions. Scc2 is not required for chromosomal association of cohesin, but Rec8 is hypo-phosphorylated in its absence. Moreover, we did not identify a homolog of the cohesin loader Scc4, and no evidence was found of auxiliary factors, such as Eco1, Pds5, or WAPL. We propose that in Tetrahymena, a single, minimal cohesin complex performs all necessary functions for germline mitosis and meiosis, but is dispensable for transcription regulation and chromatin organization of the somatic genome.

Condensin is a protein complex with diverse functions in chromatin packaging and chromosome condensation and segregation. We studied condensin in the evolutionarily distant protist model Tetrahymena, which features non-canonical nuclear organization and divisions. In Tetrahymena, the germline and soma are partitioned into two different nuclei within a single cell. Consistent with their functional specializations in sexual reproduction and gene expression, condensins of the germline nucleus and the polyploid somatic nucleus are composed of different subunits. Mitosis and meiosis of the germline nucleus and amitotic division of the somatic nucleus are all dependent on condensins. In condensin-depleted cells, a chromosome condensation defect was most striking at meiotic metaphase, when Tetrahymena chromosomes are normally most densely packaged. Live imaging of meiotic divisions in condensin-depleted cells showed repeated nuclear stretching and contraction as the chromosomes failed to separate. Condensin depletion also fundamentally altered chromosome arrangement in the polyploid somatic nucleus: multiple copies of homologous chromosomes tended to cluster, consistent with a previous model of condensin suppressing default somatic pairing. We propose that failure to form discrete chromosome territories is the common cause of the defects observed in the absence of condensins.