Biology subjects

Petry, S.

Publications and source records attributed to Petry, S..

26 records · Page 2Linked to original sources

bioRxiv2021

Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia

In Alzheimers disease (AD), hyper-phosphorylation and aggregation of tau correlates with clinical progression and represents a valid therapeutic target. A recent 20-year prospective study revealed an association between moderate to high frequency of Finnish sauna bathing and a lower incidence of dementia and AD, but the molecular mechanisms underlying these benefits remain uncertain. Here, we tested the hypothesis that sauna-like conditions could lower tau phosphorylation by increasing body temperature. We observed a decrease in tau phosphorylation in wild-type and hTau mice as well as in neuron-like cells when exposed to higher temperatures. These effects were correlated with specific changes in phosphatase and kinase activities, but not with inflammatory or heat-shock responses. We also used a drug strategy to promote thermogenesis. Topical application of menthol led to a sustained increase in body temperature in hTau mice concomitant with a significant decrease in tau phosphorylation. Our results suggest that sauna-like conditions or menthol treatment could lower tau pathology through mild hyperthermia, and may provide promising therapeutic strategies for AD and other tauopathies.

neuroscience↗

bioRxiv2020

Branching microtubule nucleation is controlled by importin-mediated inhibition of TPX2 phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed by several microtubule nucleation pathways. In the spindle center, Targeting Protein for XKlp2 (TPX2) promotes branching microtubule nucleation, where new microtubules are nucleated from pre-existing ones. Until the onset of spindle assembly, TPX2 is sequestered by importins-/{beta}, yet the molecular nature of this regulation remains unclear, particularly since TPX2 was recently found to undergo a liquid-liquid phase separation to execute its function. Here we demonstrate that TPX2 interacts with importins-/{beta} with nanomolar affinity as a 1:1:1 mono-dispersed trimer. We identify a new nuclear localization sequence (NLS) on TPX2, which contributes to its high-affinity interaction with importin-. Interestingly, importin-{beta} alone can also associate with TPX2, and does so via dispersed, weak interactions. Interactions of both importin- and importin-{beta} with TPX2 each inhibit its propensity for phase separation, and consequently its ability to orchestrate branching microtubule nucleation. In sum, our study explains how TPX2 is regulated in order to facilitate spindle assembly, and provides novel insight into how a protein phase separation can be inhibited via weak biomolecular interactions. Significance StatementThe discovery that proteins can undergo phase separation is revolutionizing biology. Characterization of dozens of phase separating proteins in vitro over the past several years has mainly focused on how macromolecules undergo liquid-liquid phase separation (LLPS). The next, and possibly bigger challenge is to investigate how LLPS is regulated in the cell, namely how it is inhibited to spatiotemporally control a certain cellular function. Here, we addressed this challenge by identifying how the spindle assembly factor TPX2 is inhibited by importins from undergoing LLPS and thereby turning on spindle assembly.

biochemistry↗

bioRxiv2020

Confinement size determines the architecture of Ran-induced microtubule networks

The organization of microtubules (MTs) within cells is critical for its internal organization during interphase and mitosis. During mitotic spindle assembly, MTs are made and organized around chromosomes in a process regulated by RanGTP. The role of RanGTP has been explored in Xenopus egg extracts, which are not limited by a cell membrane. Here, we investigated whether cell-sized confinements affect the assembly of RanGTP-induced MT networks in Xenopus egg extracts. We used microfluidics to encapsulate extract within monodisperse extract-in-oil droplets. Importantly, we find that the architecture of Ran-induced MT networks depends on the droplet diameter and the Ran concentration, and differs from structures formed in bulk extract. Our results highlight that both MT nucleation and physical confinement play critical roles in determining the spatial organization of the MT cytoskeleton. STATEMENT OF SIGNIFICANCEDuring cell division, chromosomes are segregated by the mitotic spindle, whose framework consists of up to hundreds of thousands of microtubules (MTs). Spindle MTs are generated via several pathways, one of which is regulated by RanGTP. Yet, how Ran-induced MTs self-organize within cell-sized confinement remains unclear. This work reports unexpected architectures of Ran-induced MT networks confined in cell-sized droplets, which depend on the droplet diameter and the RanGTP concentration. Thus, MT nucleation and confinement together give rise to specific MT network architectures, which are otherwise not observed in unconfined assays. The findings provide a simple strategy to engineer the architectures of MT networks and could have direct implications in nucleation-controlled soft material processing.

biophysics↗

bioRxiv2019

Molecular mechanism of microtubule nucleation from gamma-tubulin ring complex

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, {gamma}-tubulin ring complex ({gamma}-TuRC) has been identified, precisely how {gamma}-TuRC nucleates a MT remains poorly understood. Here we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis {gamma}-TuRC. We reveal a high {gamma}-/{beta}-tubulin affinity, which facilitates assembly of a MT from {gamma}-TuRC. Whereas spontaneous nucleation requires assembly of 8 {beta}-tubulins, nucleation from {gamma}-TuRC occurs efficiently with a cooperativity of 4 {beta}-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally-associated {beta}-tubulins drive {gamma}-TuRC into a closed conformation. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance {gamma}-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal {gamma}-/{beta}-tubulin interaction.

cell biology↗

bioRxiv2019

Biochemical reconstitution of branching microtubule nucleation

Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways still need to be identified along with their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-existing microtubules, which is essential for spindle assembly and chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex ({gamma}-TuRC), the two branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. Specifically, TPX2 generates regularly-spaced patches that recruit augmin and {gamma}-TuRC to microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 degrees. Our work demonstrates how {gamma}-TuRC is brought to its nucleation site for branching microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for generating a particular microtubule architecture by regulating microtubule nucleation.

cell biology↗

bioRxiv2019

Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. TPX2 is an effector of microtubule nucleation in spindles, and functions with the substrate tubulin by an unknown mechanism. Here, we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 directly demonstrate that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-/{beta} heterodimer, regulates both co-condensation and activity. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.

biochemistry↗

bioRxiv2019

Spatiotemporal organization of branched microtubule networks

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and {gamma}-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/{gamma}-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.\n\nImpact StatementA sequential reaction pathway involving TPX2, augmin and {gamma}-TuRC governs the assembly and architecture of branched microtubule networks.

biophysics↗

bioRxiv2018

Next-generation sequencing based hospital outbreak investigation yields insight into Klebsiella aerogenes population structure and determinants of carbapenem resistance and virulence

Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant K. aerogenes (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight clonal cluster (<7 SNPs apart), suggestive of a protracted intra-ward transmission event. No clonal relationships were identified between the CICU CR-KA strains and additional hospital CR-KA patient isolates from different wards and/or previous years. Genes encoding carbapenemases or drug-resistant plasmids were absent in the outbreak strains, and carbapenem resistance was attributed to mutations impacting AmpD activity and membrane permeability. The CICU outbreak strains harbored an integrative conjugative element (ICEKp10), which has been associated with pathogenicity in hypervirulent Klebsiella pneumoniae lineages. Comparative genomics with global K. aerogenes genomes showed our outbreak strains to group closely with global ST4 strains, which along with ST93 likely represent dominant K. aerogenes lineages associated with human infections. WGS is a powerful tool that goes beyond high-resolution tracking of transmission events into identifying the genetic basis of drug-resistance and virulence, which are not part of conventional diagnostic workflows. With an increasing availability of sequenced genomes from across the globe, population structure analysis offers opportunities to identify emerging trends and dominant clones associated with specific syndromes and geographical locations for poorly characterized pathogens.

genomics↗