Biology subjects

Kaplan, M.

Publications and source records attributed to Kaplan, M..

5 recordsLinked to original sources

bioRxiv2018

Rapid Tilt-Series Acquisition for Electron Cryotomography

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds (about 100x faster than current methods), but with loss of details finer than [~]4 nm. We also demonstrate a fast-incremental method that can record a tilt-series about 10x faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology.

biophysics↗

bioRxiv2018

Simulations suggest a constrictive force is required for Gram-negative bacterial cell division

To divide, Gram-negative bacterial cells must remodel their peptidoglycan cell wall to a smaller and smaller radius at the division site, but how this process occurs remains debated. While the tubulin homolog FtsZ is thought to generate a constrictive force, it has also been proposed that cell wall remodeling alone is sufficient to drive membrane constriction, possibly via a make-before-break mechanism in which new hoops of cell wall are made inside the existing hoops (make) before bonds in the existing wall are cleaved (break). Previously, we constructed software, REMODELER 1, to simulate cell wall remodeling in rod-shaped bacteria during growth. Here, we used this software as the basis for an expanded simulation system, REMODELER 2, which we used to explore different mechanistic models of cell wall division. We found that simply organizing the cell wall synthesis complexes at the midcell was not sufficient to cause wall invagination, even with the implementation of a make-before-break mechanism. Applying a constrictive force at the midcell could drive division if the force was sufficiently large to initially constrict the midcell into a compressed state before new hoops of relaxed cell wall were incorporated between existing hoops. Adding a make-before-break mechanism could drive division with a smaller constrictive force sufficient to bring the midcell peptidoglycan into a relaxed, but not necessarily compressed, state.

biophysics↗

bioRxiv2018

The structural complexity of the Gammaproteobacteria flagellar motor is related to the type of its torque-generating stators

The bacterial flagellar motor is a cell-envelope-embedded macromolecular machine that functions as a propeller to move the cell. Rather than being an invariant machine, the flagellar motor exhibits significant variability between species, allowing bacteria to adapt to, and thrive in, a wide range of environments. For instance, different torque-generating stator modules allow motors to operate in conditions with different pH and sodium concentrations and some motors are adapted to drive motility in high-viscosity environments. How such diversity evolved is unknown. Here we use electron cryo-tomography to determine the in situ macromolecular structures of the flagellar motors of three Gammaproteobacteria species: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis MR-1, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the stator system of the motor and its structural complexity. Motors with a single H+-driven stator system have only the core P- and L-rings in their periplasm; those with dual H+-driven stator systems have an extra component elaborating their P-ring; and motors with Na+- (or dual Na+-H+)- driven stator systems have additional rings surrounding both their P- and L-rings. Our results suggest an evolution of structural complexity that may have enabled pathogenic bacteria like L. pneumophila and P. aeruginosa to colonize higher-viscosity environments in animal hosts.

microbiology↗

bioRxiv2018

Stable sub-complexes observed in situ suggest a modular assembly pathway of the bacterial flagellar motor

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression, protein localization and association of a dozen or more unique components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with each subsequent component stabilizing the last. Here, using electron cryo-tomography of intact Legionella pneumophila, Pseudomonas aeruginosa and Shewanella oneidensis cells, we observe stable outer-membrane-embedded sub-complexes of the flagellar motor. These sub-complexes consist of the periplasmic embellished P- and L-rings, in the absence of other flagellar components, and bend the membrane inward dramatically. Additionally, we also observe independent inner-membrane sub-complexes consisting of the C- and MS-rings and export apparatus. These results suggest an alternate model for flagellar motor assembly in which outer- and inner-membrane-associated sub-complexes form independently and subsequently join, enabling later steps of flagellar production to proceed.

microbiology↗

bioRxiv2017

Bridging multilocus species delimitation and DNA barcoding through target enrichment of UCEs: A case study with Mexican highland frogs

Recently, molecular studies have uncovered significant cryptic diversity in the Mexican Highlands, leading to the description of many new endemic species. DNA approaches to this kind of species discovery have included both mitochondrial DNA (mtDNA) sequencing and multilocus genomic methods. While these marker types have often been pitted against one another, there are benefits to deploying them together, as linked mtDNA data can provide the bridge between uncovering lineages through rigorous multilocus genomic analysis and identifying lineages through comparison to existing mtDNA databases. Here, we apply one class of multilocus genomic marker, ultraconserved elements (UCEs), and linked mtDNA data to a species complex of frogs (Sarcohyla bistincta) found in the Mexican Highlands. We generated data from 1,891 UCEs, which contained 1,742 informative SNPs for S. bistincta and closely related species and captured mitochondrial genomes for most samples. Genetic analyses based on both whole loci and SNPs agree there are numerous distinct and divergent lineages within S. bistincta. The SNP-based species tree provides the most conservative estimate of 8 well-supported lineages in three major clades. Having linked mtDNA data allowed us to tap into the large number of mtDNA sequences available on GenBank and identify one of these lineages as an already-described species, S. pentheter. One identified clade (containing 2 of the 8 lineages) was 10% divergent in mtDNA and paraphyletic with other S. bistincta, making this clade a clear candidate for species status. Phylogenies from UCEs and mtDNA mostly agreed in their topologies, but differed in that mtDNA suggested a more complex evolutionary history perhaps influenced by gene flow between some neighboring lineages. Our study demonstrates that the Mexican Highlands still hold substantial undescribed diversity. Combining multilocus genomic data with linked mtDNA data is a useful approach for identifying potential new species and associating them with already described taxa, which is especially important in groups with undescribed subadult phenotypes, where geographic ranges are unclear, or where phenotypes are conserved.

evolutionary biology↗