Julie Delaroche's research while affiliated with Université Grenoble Alpes and other places

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

Adenomatous Polyposis Coli (APC) is a scaffold protein with tumour suppressor properties. Mutations causing the loss of its C-terminal domain (APC-C) - bearing cytoskeleton-regulating sequences - correlate with colorectal cancer. The cellular roles of APC in mitosis are widely studied, but the molecular mechanisms of its interaction with the cytoskeleton are poorly understood. Here, we investigated how APC-C regulates microtubule properties, and found it to promote both microtubule growth and shrinkage. Strikingly, APC-C accumulates at shrinking microtubule extremities, a common characteristic of depolymerases. Cryo-electron microscopy revealed that APC-C adopts an extended conformation along the protofilament crest and shows the presence of ring-like tubulin oligomers around microtubules which required the presence of two APC-C sub-domains. A mutant of APC-C incompetent to decorate microtubules with ring-like tubulin oligomers exhibited a reduced effect on microtubule dynamics. Finally, whereas native APC-C rescued defective chromosome alignment in metaphase cells silenced for APC, the ring-incompetent mutant failed to correct mitotic defects. Thus, the bilateral interaction of APC-C with tubulin and microtubules likely contributes to its mitotic functions.

Neuronal microtubules have long been known to contain intraluminal particles, called MIPs (microtubule inner proteins), most likely involved in the extreme stability of microtubules in neurons. This chapter describes a cryo-electron microscopy-based assay to visualize microtubules containing neuronal MIPs. We present two protocols to prepare MIPs-containing microtubules, using either in vitro microtubule polymerization assays or extraction of microtubules from mouse hippocampal neurons in culture.

Neuronal activities depend heavily on microtubules, which shape neuronal processes and transport myriad molecules within them. Although constantly remodeled through growth and shrinkage events, neuronal microtubules must be sufficiently stable to maintain nervous system wiring. This stability is somehow maintained by various microtubule-associated proteins (MAPs), but little is known about how these proteins work. Here, we show that MAP6, previously known to confer cold stability to microtubules, promotes growth. More unexpectedly, MAP6 localizes in the lumen of microtubules, induces the microtubules to coil into a left-handed helix, and forms apertures in the lattice, likely to relieve mechanical stress. These features have not been seen in microtubules before and could play roles in maintaining axonal width or providing flexibility in the face of compressive forces during development.

In neurons, microtubule networks alternate between single filaments and bundled arrays under the influence of effectors controlling their dynamics and organization. Tau is a microtubule bundler that stabilizes microtubules by stimulating growth and inhibiting shrinkage. The mechanisms by which tau organizes microtubule networks remain poorly understood. Here, we studied the self-organization of microtubules growing in the presence of tau isoforms and mutants. The results show that tau’s ability to induce stable microtubule bundles requires two hexapeptides located in its microtubule-binding domain and is modulated by its projection domain. Site-specific pseudophosphorylation of tau promotes distinct microtubule organizations: stable single microtubules, stable bundles, or dynamic bundles. Disease-related tau mutations increase the formation of highly dynamic bundles. Finally, cryo–electron microscopy experiments indicate that tau and its variants similarly change the microtubule lattice structure by increasing both the protofilament number and lattice defects. Overall, our results uncover novel phosphodependent mechanisms governing tau’s ability to trigger microtubule organization and reveal that disease-related modifications of tau promote specific microtubule organizations that may have a deleterious impact during neurodegeneration.

In neurons, microtubule networks alternate between single filaments and bundled arrays under the influence of effectors controlling their dynamics and organization. Tau is a microtubule bundler which stabilizes microtubules by stimulating growth and inhibiting shrinkage. The mechanisms by which tau organizes microtubule networks remain poorly understood. Here, we studied the self-organization of microtubules growing in the presence of tau isoforms and mutants. The results show that tau's ability to induce stable microtubule bundles requires two hexapeptides located in its microtubule-binding domain, and is modulated by its projection domain. Site-specific pseudo-phosphorylation of tau promotes distinct microtubule organizations: stable single microtubules, stable bundles or dynamic bundles. Disease-related tau mutations increase the formation of highly dynamic bundles. Finally, cryo-electron microscopy experiments indicate that tau and its variants similarly change the microtubule lattice structure by increasing both the protofilament number and lattice defects. Overall, our results uncover novel phospho-dependent mechanisms governing tau's ability to trigger microtubule organization and reveal that disease-related modifications of tau promote specific microtubule organizations which may have a deleterious impact during neurodegeneration.

Tau is a major microtubule-associated protein (MAP) mainly expressed in the brain. Tau binds the lattice of microtubules and favors their elongation and bundling. Recent studies have shown that tau is also a partner of end-binding proteins (EBs) in neurons. EBs belong to the protein family of the plus-end tracking proteins that preferentially associate with the growing plus-ends of microtubules and control microtubule end behavior and anchorage to intracellular organelles. Reconstituted cell-free systems using purified proteins are required to understand the precise mechanisms by which tau influences EB localization on microtubules and how the concerted activity of these two MAPs modulates microtubule dynamics. We developed an in vitro assay combining TIRF microscopy and site-directed mutagenesis to dissect the interaction of tau with EBs and to study how this interaction affects microtubule dynamics. Here, we describe the detailed procedures to purify proteins (tubulin, tau, and EBs), prepare the samples for TIRF microscopy, and analyze microtubule dynamics, and EB binding at microtubule ends in the presence of tau.

This study evaluates the extravasation pathways of circulating macromolecules in a rat glioma model (RG2) which was observed by both magnetic resonance imaging using ultrasmall superparamagnetic iron oxide and electron microscopy. Although magnetic resonance imaging signal enhancement was observed as soon as 10 min after injection (9.4% 2 h after injection), electron microscopy showed that endothelial cells were still tightly sealed. However, circulating immunoglobulin G and ultrasmall superparamagnetic iron oxide were found in large membrane compartments of endothelial cells, in the basal lamina (7.4 ± 1.2 gold particles/µm2 in the tumor versus 0.38 ± 0.17 in healthy tissue, p = 1.4.10−5) and between tumoral cells. Altogether, this strongly suggests an active transport mediated by macropinocytosis. To challenge this transport mechanism, additional rats were treated with amiloride, an inhibitor of macropinocytosis, leading to a reduction of membrane protrusions (66%) and of macropinosomes. Amiloride however also opened tumoral tight junctions allowing a larger extravasation of ultrasmall superparamagnetic iron oxide (magnetic resonance imaging signal enhancement of 35.7% 2 h after injection). Altogether, these results suggest that ultrasmall superparamagnetic iron oxide and immunoglobulin G in the RG2 glioma model follow an active extravasation pathway mediated by a macropinocytosis process. Amiloride also appears as a potential strategy to facilitate the extravasation of chemotherapeutic drugs in glioma.