Biology subjects

Prowse, E. N. P.

Publications and source records attributed to Prowse, E. N. P..

2 recordsLinked to original sources

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↗

bioRxiv2022

Huntingtin S421 phosphorylation increases kinesin and dynein engagement on early endosomes and lysosomes

Huntingtin (HTT) is a scaffolding protein that recruits motor proteins to vesicular cargoes, enabling it to regulate kinesin-1, dynein, and myosin-VI-dependent transport. To maintain the native stoichiometry of huntingtin with its interacting partners, we used CRISPR/Cas9 to induce a phosphomimetic mutation of the endogenous HTT at S421 (HTT-S421D). Using single particle tracking, optical tweezers, and immunofluorescence, we examined the effects of this mutation on the motility of early endosomes and lysosomes. In HTT-S421D cells, lysosomes exhibit longer displacements and higher processive fractions compared to wild-type (HTT-WT) cells. Kinesins and dyneins exert greater forces on early endosomes and lysosomes in cells expressing HTT-S421D. Additionally, endosomes bind to microtubules faster and are more resistant to detachment under load. The recruitment of kinesins and dyneins to microtubules is enhanced in HTT-S421D cells. In contrast, overexpression of HTT had variable effects on the processivity, displacement, and directional bias of both early endosomes and lysosomes. These data indicate that phosphorylation of the endogenous huntingtin causes early endosomes and lysosomes to move longer distances and more processively by recruiting and activating both kinesin-1 and dynein. Statement of SignificanceThe ubiquitous scaffolding protein huntingtin regulates the recruitment and activity of microtubule motors. Huntingtin phosphorylation at S421 enhances the microtubule binding and force generation of kinesin and dynein on early endosomes and lysosomes. Using optical tweezers to measure the forces exerted on endosomes in CRISPR-engineered cells, we find that a phosphomimetic huntingtin mutation (S421D) enhances both kinesin- and dynein-driven forces on early endosomes and lysosomes. The ability to modulate motor activity on a range of organelles makes huntingtin unique and suggests a significant role for huntingtin in regulating intracellular transport.

biophysics↗