Biology subjects

Cho, M.

Publications and source records attributed to Cho, M..

4 recordsLinked to original sources

bioRxiv2018

Structural basis of ECF-sigma-factor-dependent transcription initiation.

Extracytoplasmic (ECF) {sigma} factors, the largest class of alternative {sigma} factors, are related to primary {sigma} factors, but have simpler structures, comprising only two of the six conserved functional modules present in primary {sigma} factors: region 2 ({sigma}R2) and region 4 ({sigma}R4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF {sigma} factor {sigma}L, and promoter DNA. The structures show that {sigma}R2 and {sigma}R4 of the ECF {sigma} factor occupy the same sites on RNAP as in primary {sigma} factors, show that the connector between {sigma}R2 and {sigma}R4 of the ECF {sigma} factor--although unrelated in sequence--follows the same path through RNAP as in primary {sigma} factors, and show that the ECF {sigma} factor uses the same strategy to bind and unwind promoter DNA as primary {sigma} factors. The results define protein-protein and protein-DNA interactions involved in ECF-{sigma}-factor-dependent transcription initiation.

molecular biology↗

bioRxiv2018

The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans

The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.

microbiology↗

bioRxiv2017

An Observational Study of Ballooning in Large Spiders: Nanoscale Multi-Fibres Enable Large Spiders’ Soaring Flight

The physical mechanism of aerial dispersal of spiders, \"ballooning behavior,\" is still unclear because of the lack of serious scientific observations and experiments. Therefore, as a first step in clarifying the phenomenon, we studied the ballooning behavior of relatively large spiders (heavier than 5 mg) in nature. Additional wind tunnel tests to identify ballooning silks were implemented in the laboratory. From our observation, it seems obvious that spiders actively evaluate the condition of the wind with their front leg (leg I) and wait for the preferable wind condition for their ballooning takeoff. In the wind tunnel tests, as yet unknown physical properties of ballooning fibers (length, thickness and number of fibers) were identified. Large spiders, 16-20 mg Xysticus species, spun 50 to 60 nanoscale fibers, with a diameter of 121 to 323 nm. The length of these threads was 3.22 {+/-} 1.31 m (N = 22). These physical properties of ballooning fibers can explain the ballooning of large spiders with relatively light updrafts, 0.1-0.5 m s-1, which exist in a light breeze of 1.5-3.3 m s-1. Additionally, in line with previous research on turbulence in atmospheric boundary layers and from our wind measurements, it is hypothesized that spiders use the ascending air current for their aerial dispersal, the \"ejection\" regime, which is induced by hairpin vortices in the atmospheric boundary layer turbulence. This regime is highly correlated with lower wind speeds. This coincides well with the fact that spiders usually balloon when the wind speed is lower than 3 m s-1.

biophysics↗

bioRxiv2017

Correction of autoimmune IL2RA mutations in primary human T cells using non-viral genome targeting

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2-5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

genetics↗