Biology subjects

Brouhard, G. J.

Publications and source records attributed to Brouhard, G. J..

5 recordsLinked to original sources

bioRxiv2025

Competition for microtubule lattice spacing between a microtubule expander and compactor.

Microtubules exist in expanded and compacted states, as defined by the lattice spacing of {beta}-tubulin dimers. Changes in lattice spacing has been linked to factors such as GTP-hydrolysis, the binding of microtubule-associated proteins (MAPs), the tubulin code, and microtubule bending. These diverse factors exert opposing molecular driving forces on the microtubule lattice that push lattice spacing towards expanded or compacted states. To better understand how these opposing forces are reconciled, we developed in vitro and cell-based model systems for the competition between a microtubule expander (paclitaxel) and a microtubule compactor (Doublecortin, or DCX). Using an in vitro reconstitution approach, we show that paclitaxel expands microtubules cooperatively. In cells, high concentrations of paclitaxel cause DCX to relocalize to compacted lattices found at concave bends. When the concentration of DCX is increased, however, we find that DCX re-compacts the previously expanded microtubules in vitro. Consistently, high expression levels of DCX prevent its relocalization in paclitaxel treated cells. When the competition between paclitaxel and DCX is "balanced", we observe a complex phenotype: DCX simultaneously localized to both long, straight clusters and concave bends, while other regions on the microtubule network remained DCX-free. We conclude that multiple lattice spacings can coexist in cells. Our results indicate that competition for microtubule lattice spacing is a critical aspect of microtubule physiology.

cell biology↗

bioRxiv2024

Huntingtin polyglutamine expansions misdirect axonal transport by perturbing motor and adaptor recruitment

Huntingtons disease (HD) is caused by polyglutamine (polyQ) expansions in huntingtin (HTT). Polyglutamine repeat lengths >35Q lead to neurodegeneration and longer repeats correspond to earlier symptom onset. HTT scaffolds kinesin-1 and dynein to a variety of vesicles and organelles directly and through adaptors. To characterize the effects of HTT polyQ expansions on axonal transport, we tracked BDNF vesicles, mitochondria, and lysosomes in neurons induced from an isogenic set of human stem cell lines with repeat lengths of 30, 45, 65, and 81Q. Mild and intermediate pathogenic polyQ expansions caused increased BDNF motility, while HTT-81Q misdirected BDNF towards the distal tip. In comparison, mitochondria and lysosome transport showed mild defects with polyQHTT. We next examined the effect of polyQHTT in combination with neuroinflammatory stress. Under stress, BDNF cargoes in HTT-30Q neurons were more processive. Stress in HTT-81Q resulted in a stark decrease in the number of BDNF cargoes. However, the few remaining BDNF cargoes displayed more frequent long-range motility in both directions. Under neuroinflammatory stress, lysosomes were more abundant in HTT-81Q neurons, and motile lysosomes moved less processively and had an anterograde bias while lysosomes in HTT-30Q where not strongly affected. To examine how HTT-polyQ expansions altered the motors and adaptors on vesicular cargoes, we isolated BDNF cargoes from neurons and quantified the proteins associated with them. BDNF-endosomes isolated from HTT-81Q neurons associated with 2.5 kinesin-1 and 3.9 HAP1 molecules on average, compared to 1.0 kinesin-1 and 1.0 HAP1 molecule for HTT-30Q neurons. Together, these results show that polyQ expansions in HTT cause aberrant motor and adaptor recruitment to cargoes, resulting in dysregulated transport and responses to neuroinflammatory stress.

cell biology↗

bioRxiv2024

Microtubule perturbations of neurite extension and nucleokinesis in an iPSC-derived model system

In neurons, patterns of different microtubule types are essential for neurite extension and nucleokinesis. Cellular model systems such as rodent primary cultures and induced pluripotent stem cells (iPSC)-derived neurons have provided key insights into how these patterns are created and maintained through the action of microtubule-associated proteins (MAPs), motor proteins, and regulatory enzymes. iPSC-derived models show tremendous promise but lack benchmarking and validation relative to rodent primary cultures. Here we have characterized a recent iPSC-derived model, in which doxycycline-induced expression of Neurogenin-2 drives consistent trans-differentiation into the neuronal state (EBiSC-NEUR1 neurons, referred to as NGN2 neurons below). We developed a suite of open-access, semi-automated methods to measure neurite extension and nucleokinesis of NGN2 neurons, which compare favorably to published data from other models. Then, we challenged NGN2 neurons with a panel of drugs that perturb microtubule physiology. NGN2 neurons extension and nucle-okinesis were significantly perturbed by two microtubule-targeting drugs, namely a taxane (paclitaxel) and a vinca alkaloid (DZ-2384). In contrast, inhibition of microtubule severing (spastazoline) or of deacetylation (trichostatin A) had a limited effect on nucleokinesis only. Our results support the primary importance of microtubule dynamics in neuronal development and demonstrate the power of NGN2 neurons as a model system.

cell biology↗

bioRxiv2023

Doublecortin regulates neuronal migration by editing the tubulin code

Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) that binds directly to microtubules via two Doublecortin (DC) domains. The DC domains sense the nucleotide state, longitudinal curvature, and protofilament number of the microtubule lattice, indicating a role in the regulation of microtubule structure in neurons. Mutations in DCX cause lissencephaly and subcortical band heterotopia (also known as double-cortex syndrome) due to impaired neuronal migration. To better understand the role of DCX in neuronal migration, we developed a model system based on induced pluripotent stem cells (iPSCs). We used CRISPR/Cas9 to knock-out the Dcx gene in iPSCs and differentiated the cells into cortical neurons. Compared to control neurons, the DCX-KO neurons showed reduced velocities of nuclear movements. The reduced velocities coincided with an increase in the number of neurites early in neuronal development, consistent with a neuronal migration phenotype and previous findings in a DCX-KO mouse model. Neurite branching is regulated by a host of MAPs and other factors, as well as by microtubule polymerization dynamics. However, EB comet dynamics were unchanged in DCX-KO neurons, with similar growth rates, lifetimes, and numbers. Rather, we observed a significant reduction in -tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching were rescued by expression of DCX or of TTLL11, an -tubulin glutamylase. Using U2OS cells as an orthogonal model system, we show that DCX and TTLL11 act synergistically to promote polyglutamylation. Polyglutamylation regulates numerous MAPs, severing enzymes, and molecular motors. Consistently, we observe that lysosomes in DCX-KO neurons show a reduction of their processivity. We propose that the DCX acts as a positive regulator of -tubulin polyglutamylation and restricts neurite branching. Our results indicate an unexpected role for DCX in the homeostasis of the tubulin code.

cell biology↗

bioRxiv2018

Numerical parameter space compression and its application to microtubule dynamic instability

Physical models of biological systems can become difficult to interpret when they have a large number of parameters. But the models themselves actually depend on (i.e. are sensitive to) only a subset of those parameters. Rigorously identifying this subset of \"stiff\" parameters has been made possible by the development of parameter space compression (PSC). However, PSC has only been applied to analytically-solvable physical models. We have generalized this powerful method by developing a numerical approach to PSC that can be applied to any computational model. We validated our method against analytically-solvable models of random walk with drift and protein production and degradation. We then applied our method to an active area of biophysics research, namely to a simple computational model of microtubule dynamic instability. Such models have become increasingly complex, perhaps unnecessarily. By adding two new parameters that account for prominent structural features of microtubules, we identify one that can be \"compressed away\" (the \"seam\" in the microtubule) and another that is essential to model performance (the \"tapering\" of microtubule ends). Furthermore, we show that the microtubule model has an underlying, low-dimensional structure that explains the vast majority of our experimental data. We argue that numerical PSC can identify the low-dimensional structure of any computational model in biophysics. The low-dimensional structure of a model is easier to interpret and identifies the mechanisms and experiments that best characterize the system.

biophysics↗