Biology subjects

Castillo, J. P.

Publications and source records attributed to Castillo, J. P..

6 recordsLinked to original sources

bioRxiv2025

Tuning the gate and the gear: The LRRC26 (γ1) subunit modulates intrinsic gating and voltage-sensor coupling of the BK channel

Association of auxiliary subunits ({beta}1-4 and {gamma}1-4) with the pore-forming subunit of the calcium- and voltage-activated potassium (BK) channel provides functional diversity. {gamma}1 promotes a significant leftward shift of the voltage activation curve, ensuring the adequate functioning of secretory glands, allowing the BK channel to release K+ at the cells resting Ca2+ concentration. Given its physiological importance, it is crucial to elucidate the mechanisms of {gamma}1 action. However, structural and functional studies have yielded conflicting conclusions regarding the modulation of BK channels by {gamma}1. Here, using macroscopic, single-channel, and gating current measurements, we demonstrate that at zero mV {gamma}1 increases 92-fold the equilibrium constant that defines the closed- open transition by destabilizing the channels closed configuration and enhancing the coupling between the voltage sensor and the pore domain, without affecting voltage-sensor activation. These results suggest that {gamma}1 not only causes an increase in the energetic coupling between the voltage sensors and the pore but mainly enhances the channel opening reaction. TeaserThe {gamma}1 subunit favors the BK channel pore opening by destabilizing its closed configuration.

biophysics↗

bioRxiv2024

Interspecies relationships of wild amoebae and bacteria with C. elegans create environments propitious for multigenerational diapause.

The molecular and physical communication within the microworld supports the entire web of life as we know it. How organisms such as bacteria, amoeba and nematodes -all superabundant-interact to sustain their niche, however, is not known, especially how their associations generate and affect behavior of animals in fluctuating environments. To have a frame to study interactions between microbe and animal, we collected soil from a temperate semi-arid climate and isolated the culturable genus of bacteria Comamonas, Stenotrophomonas, Chryseobacterium and Rhodococcus and the amoeba Tetramitus. This ensemble was then fed in long-term experiments to the nematode C. elegans to study developmental rate, diapause entry, fertility, feeding behavior and neuronal integrity. We observed that the ensemble is long lasting and induces animals to diapause after a few generations under conditions that are not canonically pathogenic. We called this phenomenon Dauer Formation in Natural Ensembles (DaFNE). DaFNE requires the communication between live bacteria and the nematode intestine, suggesting the existence of a bidirectional interaction in the holobiont. While all bacteria from the ensemble colonize the intestine of the nematodes, Comamonas is the most represented and Rhodococcus the scarcest. The amoeba Tetramitus can be ingested by C. elegans, but it is not part of its microbiota. DaFNE depends on pheromone and nematode quorum, but high temperature in the homeostatic range, triggers diapause with fewer numbers. DaFNE increases as generations pass and is also remembered transgenerationally. The RNA interference (RNAi) pathway is needed for initiation of DaFNE, indicating the communication via RNA is crucial to execute bacterially induced behaviors in natural environments. SignificanceMicrobes have an overwhelming influence over the animals they live with, modulating development and decision making. Microscopic nematodes are the most abundant multicellular animals in the biosphere, suggesting they possess well-rehearsed successful relationships with their associated microbiota. Little is known about the modulation of nematode behavior in complex ecosystems with multiple organisms interacting. We use bacteria and amoeba from a natural ecosystem and introduce the pioneer nematode C. elegans to study behavioral parameters in long lasting experiments. The most striking response of nematodes to this natural environment is the commitment to diapause of a significant portion of the population. We call this form of hibernation Dauer Formation in Natural Ensembles or DaFNE. We propose that animals in nature may hibernate frequently, as a result of the communication with their natural biota. We find that DaFNE requires pheromone production in nematodes and also the RNA interference pathway, suggesting the RNA repertoire of both entities may be at play. Higher temperatures in the optimal range for nematode growth, require much less nematode quorum for DaFNE, indicating that a non-noxious increase in temperature favors diapause in natural environments. Nematodes respond to each bacterium in different ways when grown in monocultures and in the ensemble. This suggests that the abundance of specific species in nature may shift behavioral preferences and outputs in microscopic animals. We also show that the amoeba Tetramitus can be ingested by worms, demonstrating that C. elegans is a broader microbivore. Like worms, amoebae display specific responses to bacteria and add variability to behaviors elicited by nematodes. Finally, bacteria in the ensemble unlike in monocultures, are not exhausted during the length of the experiments even in the presence of bacterivore nematodes and amoebae.

microbiology↗

bioRxiv2022

The assembly of wild natural isolates define neuronal integrity and life history traits of co-inhabiting C. elegans.

Bacterivore nematodes are the most abundant animals in the biosphere, largely contributing to global biogeochemistry. The effect of environmental microbes as source of associated microbiota and natural diet on their life history traits of nematodes is likely to impact the general health of the biosphere. Caenorhabditis elegans is a unique model to study the behavioral and physiological outputs of different available microbial diets. Nonetheless, most studies are on monoaxenic cultures of laboratory bacteria while the effect of natural microbiota isolates has only recently started to be reported. Here, we quantified physiological, phenotypical and behavioral traits of worms feeding on two bacteria that co-isolated with wild nematodes and tested how combinations of these isolates with other bacteria affected the traits measured. These bacteria were identified as a putative novel species of Stenotrophomonas denominated Stenotrophomonas sp. Iso1 and a strain of Bacillus pumilus designated Iso2. The isolates induced distinctive behaviors and development patterns that changed in mixes of the two bacteria and/or the pathogen Salmonella enterica. Focusing on the degeneration rate of the touch circuit of C. elegans we show that B. pumilus alone is protective while the mix with Stenotrophomonas sp. is degenerative. The analysis of the metabolite content of each isolate and their combination identified NAD+ as potentially neuroprotective. In vivo supplementation shows that NAD+ restores neuroprotection to the mixes and also to individual non-protective bacteria. The results highlight the need to study the physiological effects of bacteria resembling native diets in a multicomponent scenario rather than using single isolates. ImportanceThe behavioral decisions of animals depend on their microbiota. In nature it is unknown how this interaction affects the health of the biosphere. To study how the nematode-bacteria relationship impacts the life history traits of these animals, we isolated bacteria found in association with wild nematodes and tested their influence as single species and consortia, in the life history traits of the model C. elegans. We identify metabolites from wild bacteria that change these traits. The bacteria isolated were identified a Stenotrophomonas sp and a B. pumilus. We find that all traits depend on the biota composition. For example, B. pumilus is neuroprotective to degenerating neurons of the touch circuit of C. elegans needed to sense and escape from predators in the wild. The co-culture with Stenotrophomonas sp. eliminates the protection. We identified NAD+ as the metabolite lost in the mix, and show that NAD+ by itself is neuroprotective.

microbiology↗

bioRxiv2022

Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetrical occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection we show that the low-force transition corresponds to the undoing of the outer-wrap of one side of the nucleosome (~27 bp), a process that can occur either cooperatively or non-cooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ~76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition, and another that coincides but does not contribute to the high-force transition. Significance statementNucleosomes, the fundamental structural unit of chromatin, consists of ~147 DNA base pairs wrapped around a histone protein octamer. Determining the forces required to unwrap the DNA from the core particle and the stepwise transitions involved in the process are essential to characterize the strength of the nucleosomal barrier and its contribution as a mechanism of control of gene expression. Here, we performed combined optical tweezers and single-molecule fluorescence measurements to annotate the specific DNA segments unwrapping during the force transitions observed in mechanical unwrapping of nucleosomes. Furthermore, we characterize the mechanical signatures of subnucleosomal particles: hexasomes and tetrasomes. The characterization performed in this work is essential for the interpretation of ongoing studies of chromatin remodelers, polymerases, and histone chaperones.

biophysics↗

bioRxiv2022

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ( reverse-topology), but they can also be directed towards the cytosol ( normal-topology). Of the ESCRT complexes 0-III, ESCRT-III is most directly implicated in membrane severing. Various subunits of ESCRT-III recruit the AAA+ ATPase VPS4, which is essential for ESCRT disassembly and reverse topology membrane scission. The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing, perhaps in conjunction with a AAA+ ATPase. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal topology membrane scission. In order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin, we sought to reconstitute the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles (GUVs) using an optical trap. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1-scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. Spastin colocalized with CHMP1B enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 but did not lead to scission. These data show that the CHMP1B and IST1 tubular coat contributes to membrane scission. Constriction alone is insufficient for scission. However, the dynamical extension of the coated tube does lead to scission. Finally, we find that in the normal topology setting analyzed here, scission is independent of VPS4 and spastin. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism.

biophysics↗

bioRxiv2020

A DNA translocase operates by cycling between planarand lock-washer structures

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport substrates possessing a preexisting helical structure, such as the bacteriophage {phi}29 dsDNA packaging motor. During each cycle, this pentameric motor tracks one helix strand (the tracking strand), and alternates between two segregated phases: a dwell in which it exchanges ADP for ATP and a burst in which it packages a full turn of DNA in four steps. We challenge this motor with DNA-RNA hybrids and dsRNA substrates and find that it adapts the size of its burst to the corresponding shorter helical pitches by keeping three of its power strokes invariant while shortening the fourth. Intermittently, the motor loses grip when the tracking strand is RNA, indicating that it makes load-bearing contacts with the substrate that are optimal with dsDNA. The motor possesses weaker grip when ADP-bound at the end of the burst. To rationalize all these observations, we propose a helical inchworm translocation mechanism in which the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

biophysics↗