Biology subjects

Castle, B. T.

Publications and source records attributed to Castle, B. T..

4 recordsLinked to original sources

bioRxiv2025

UNC-45A Drives ATP-Independent Microtubule Destabilization via Defect Recognition and Repair Inhibition Contributing to Neurite Dystrophy

UNC-45A is the only known ATP-independent microtubule severing protein. Using in vitro reconstitution and TIRF microscopy, we show that, unlike canonical severing enzymes such as katanin, spastin, and fidgetin, which hydrolyze ATP to remove tubulin dimers and promote lattice repair, UNC-45A selectively binds to pre-existing microtubule defects and inhibits GTP-tubulin incorporation. This mechanism prevents the formation of stabilized hot spots that typically protect microtubules from disassembly, resulting in persistent lattice damage and net microtubule loss, even in the presence of physiological levels of free GTP-tubulin. We further demonstrate that UNC-45A localizes near amyloid deposits in both mouse models and human cases of Alzheimers disease (AD). In cultured neurons, UNC-45A accumulates in axonal swellings--regions of pronounced microtubule disruption and experimental surrogates for dystrophic neurites in AD--and exacerbates their size and number, particularly under conditions mimicking microtubule damage. Notably, this is the first report of a microtubule severing protein that both localizes near amyloid plaques in tissue and accumulates in neurite swellings in cultured neurons, where it modulates their pathology. Together, our findings establish the mechanism of ATP-independent, damage-responsive severing pathway that couple defects recognition to repair inhibition, defining a new paradigm in microtubule quality control with broad implications for cytoskeletal integrity and remodeling in health and disease.

cell biology↗

bioRxiv2020

Biophysical modeling of the SARS-CoV-2 viral cycle reveals ideal antiviral targets

Effective therapies for COVID-19 are urgently needed. Presently there are more than 800 COVID-19 clinical trials globally, many with drug combinations, resulting in an empirical process with an enormous number of possible combinations. To identify the most promising potential therapies, we developed a biophysical model for the SARS-CoV-2 viral cycle and performed a sensitivity analysis for individual model parameters and all possible pairwise parameter changes (162 = 256 possibilities). We found that model-predicted virion production is fairly insensitive to changes in most viral entry, assembly, and release parameters, but highly sensitive to some viral transcription and translation parameters. Furthermore, we found a cooperative benefit to pairwise targeting of transcription and translation, predicting that combined targeting of these processes will be especially effective in inhibiting viral production.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

Tau avoids the GTP cap at growing microtubule plus ends

Plus-end tracking proteins (+TIPs) are a group of proteins that associate with the growing end of microtubules and mediate important cellular functions including neural development and cell division. Work in recent years has shown that the majority of +TIPs are directed to the plus-end through a family of end binding proteins (EBs), which preferentially bind the stabilizing cap of GTP-tubulin present during microtubule growth, versus weaker binding to GDP-tubulin in the proximal microtubule. One question yet to be addressed is whether there may exist other microtubule associated proteins (MAPs) that preferentially bind specific nucleotide states of tubulin. Here we report that the neuronal MAP tau, which is enriched in axons where it promotes microtubule growth and bundling, preferentially binds GDP-tubulin (KD = 0.26 {micro}M) over GMPCPP-tubulin (KD = 1.1 {micro}M) in vitro as well as GTP-tubulin at the tips of growing microtubules in vitro and in vivo. This nucleotide preference causes tau binding to lag behind the growing microtubule plus-end by about 100-200 nm both in vitro and in live cells. Thus, tau is a microtubule tip avoiding protein, establishing a new class of tip avoiding MAPs, and acts primarily by suppressing microtubule shortening rather than promoting growth. We speculate that neurological disease-relevant tau mutations may exert their phenotype by their failure to properly recognize GDP-tubulin, thus displacing +TIPs, such as EB3, and their associated activities into abnormal locations in the neuron.

cell biology↗

bioRxiv2019

Multi-scale Computational Modeling of Tubulin-Tubulin Lateral Interaction

Microtubules are multi-stranded polymers in eukaryotic cells that support key cellular functions such as chromosome segregation, motor-based cargo transport, and maintenance of cell polarity. Microtubules self-assemble via \"dynamic instability,\" where the dynamic plus ends switch stochastically between alternating phases of polymerization and depolymerization. A key question in the field is what are the atomistic origins of this switching, i.e. what is different between the GTP- and GDP-tubulin states that enables microtubule growth and shortening, respectively? More generally, a major challenge in biology is how to connect theoretical frameworks across length-time scales, from atoms to cellular behavior. In this study, we describe a multi-scale model by linking atomistic molecular dynamics (MD), molecular Brownian dynamics (BD), and cellular-level thermo-kinetic (TK) modeling of microtubules. Here we investigated the underlying interaction energy landscape when tubulin dimers associate laterally by performing all-atom molecular dynamics simulations. We found that the lateral free energy is not significantly different among three nucleotide states of tubulin, GTP, GDP, and GMPCPP, and is estimated to be {cong}-11 kBT. Furthermore, using MD potential energy in our BD simulations of tubulin dimers in solution confirms that the lateral bond is weak on its own with a mean lifetime of ~0.1 s, implying that the longitudinal bond is required for microtubule assembly. We conclude that nucleotide-dependent lateral bond strength is not the key mediator microtubule dynamic instability, implying that GTP acts elsewhere to exert its stabilizing influence on microtubule polymer. Furthermore the estimated bond strength is well-aligned with earlier estimates based on thermokinetic (TK) modeling and light microscopy measurements (VanBuren et al., PNAS, 2002). Thus, we have computationally connected atomistic level structural information, obtained by cryo-electron microscopy, to cellular scale microtubule assembly dynamics using a combination of MD, BD, and TK models to bridge from [A]ngstroms to micrometers and from femtoseconds to minutes.

biophysics↗