Competition between physical search and a weak-to-strong transition rate-limits kinesin binding times.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions. In this work, we leverage theory and experiments to identify and quantify the rate-limiting mechanism of the initial association between a cargo-bound kinesin motor and a microtubule track. Recent advances in optical tweezers provide binding times for several lengths of kinesin motors trapped at varying distances from a microtubule, empowering the investigation of competing models. We first explore a diffusion-limited model of binding. Through Brownian dynamics simulations and simulation-based inference, we find this simple diffusion model fails to explain the experimental binding times, but an extended model that accounts for the ADP state of the molecular motor agrees closely with the data, even under the scrutiny of penalizing for additional model complexity. We provide quantification of both kinetic rates and biophysical parameters underlying the proposed binding process. Our model suggests that a typical binding event is limited by ADP state rather than physical search. Lastly, we predict how these association rates can be modulated in distinct ways through variation of environmental concentrations and physical properties.

A new method to experimentally quantify dynamics of initial protein-protein interactions.

Cells run on initiation of protein-protein interactions, which are dynamically tuned spatially and temporally to modulate cellular events. This tuning can be physical, such as attaching the protein to a cargo or protein complex, thereby altering its diffusive properties, or modulating the distance between protein pairs, or chemical, by altering the proteins' conformations (e.g., nucleotide binding state of an enzyme, post-translational modification of a protein, etc.). Because a dynamic and changing subset of proteins in the cell could be in any specific state, ensemble measurements are not ideal-to untangle which of the factors are important, and how, we need single-molecule measurements. Experimentally, until now we have not had good tools to precisely measure initiation of such protein-protein interactions at the single-molecule level. Here, we develop a new method to measure dynamics of initial protein-protein interactions, allowing measurement of how properties such as the distance between proteins, and their tethered length can modulate the rate of interactions. In addition to precise measurement distance dependent motor-MT rebinding dynamics, we demonstrate the use of a dithered optical trap to measure dynamic motor-MT interactions and further discuss the possibilities of this technique being applicable to other systems.

Two-pore channel 2 is required for soluble adenylyl cyclase-dependent regulation of melanosomal pH and melanin synthesis.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

Epigenetic regulation by TET1 in gene-environmental interactions influencing susceptibility to congenital malformations.

The etiology of neural tube defects (NTDs) involves complex gene-environmental interactions. Folic acid (FA) prevents NTDs, but the mechanisms remain poorly understood and at least 30% of human NTDs resist the beneficial effects of FA supplementation. Here, we identify the DNA demethylase TET1 as a nexus of folate-dependent one-carbon metabolism and genetic risk factors post-neural tube closure. We determine that cranial NTDs in Tet1 -/- embryos occur at two to three times higher penetrance in genetically heterogeneous than in homogeneous genetic backgrounds, suggesting a strong impact of genetic modifiers on phenotypic expression. Quantitative trait locus mapping identified a strong NTD risk locus in the 129S6 strain, which harbors missense and modifier variants at genes implicated in intracellular endocytic trafficking and developmental signaling. NTDs across Tet1 -/- strains are resistant to FA supplementation. However, both excess and depleted maternal FA diets modify the impact of Tet1 loss on offspring DNA methylation primarily at neurodevelopmental loci. FA deficiency reveals susceptibility to NTD and other structural brain defects due to haploinsufficiency of Tet1. In contrast, excess FA in Tet1 -/- embryos drives promoter DNA hypermethylation and reduced expression of multiple membrane solute transporters, including a FA transporter, accompanied by loss of phospholipid metabolites. Overall, our study unravels interactions between modified maternal FA status, Tet1 gene dosage and genetic backgrounds that impact neurotransmitter functions, cellular methylation and individual susceptibilities to congenital malformations, further implicating that epigenetic dysregulation may underlie NTDs resistant to FA supplementation.

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

The spore-forming intestinal pathogen Clostridioides difficile causes multidrug resistant infection with a high rate of recurrence after treatment. Piscidins 1 (p1) and 3 (p3), cationic host defense peptides with micromolar cytotoxicity against C. difficile, sensitize C. difficile to clinically relevant antibiotics tested at sublethal concentrations. Both peptides bind to Cu2+ using an amino terminal copper and nickel binding motif. Here, we investigate the two peptides in the apo and holo states as antibiotic adjuvants against an epidemic strain of C. difficile. We find that the presence of the peptides leads to lower doses of metronidazole, vancomycin, and fidaxomicin to kill C. difficile. The activity of metronidazole, which targets DNA, is enhanced by a factor of 32 when combined with p3, previously shown to bind and condense DNA. Conversely, the activity of vancomycin, which acts at bacterial cell walls, is enhanced 64-fold when combined with membrane-active p1-Cu2+. As shown through microscopy monitoring the permeabilization of membranes of C. difficile cells and vesicle mimics of their membranes, the adjuvant effect of p1 and p3 in the apo and holo states is consistent with a mechanism of action where the peptides enable greater antibiotic penetration through the cell membrane to increase their bioavailability. The variations in effects obtained with the different forms of the peptides reveal that while all piscidins generally sensitize C. difficile to antibiotics, co-treatments can be optimized in accordance with the underlying mechanism of action of the peptides and antibiotics. Overall, this study highlights the potential of antimicrobial peptides as antibiotic adjuvants to increase the lethality of currently approved antibiotic dosages, reducing the risk of incomplete treatments and ensuing drug resistance.

Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of structurally identified and yet-undefined metabolites across tissue cryosections. While numerous software packages enable pixel-by-pixel imaging of individual metabolites, the research community lacks a discovery tool that images all metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs informs discovery of unanticipated molecules contributing to shared metabolic pathways, uncovers hidden metabolic heterogeneity across cells and tissue subregions, and indicates single-timepoint flux through pathways of interest. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling and instrument drift, markedly enhances spatial image resolution, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent tool to enhance knowledge obtained from current spatial metabolite profiling technologies.

Secretome profiling reveals acute changes in oxidative stress, brain homeostasis, and coagulation following short-duration spaceflight.

As spaceflight becomes more common with commercial crews, blood-based measures of crew health can guide both astronaut biomedicine and countermeasures. By profiling plasma proteins, metabolites, and extracellular vesicles/particles (EVPs) from the SpaceX Inspiration4 crew, we generated "spaceflight secretome profiles," which showed significant differences in coagulation, oxidative stress, and brain-enriched proteins. While >93% of differentially abundant proteins (DAPs) in vesicles and metabolites recovered within six months, the majority (73%) of plasma DAPs were still perturbed post-flight. Moreover, these proteomic alterations correlated better with peripheral blood mononuclear cells than whole blood, suggesting that immune cells contribute more DAPs than erythrocytes. Finally, to discern possible mechanisms leading to brain-enriched protein detection and blood-brain barrier (BBB) disruption, we examined protein changes in dissected brains of spaceflight mice, which showed increases in PECAM-1, a marker of BBB integrity. These data highlight how even short-duration spaceflight can disrupt human and murine physiology and identify spaceflight biomarkers that can guide countermeasure development.