Vimentin undergoes liquid–liquid phase separation to form droplets which wet and stabilize actin fibers

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(10)

Published: March 3, 2025

The cytoskeleton is composed of F-actin, microtubules, and intermediate filaments (IFs). Vimentin one the most ubiquitous well-studied IFs. It involved in many activities including wound healing, tissue fibrosis, cancer metastasis, all which require rapid vimentin IF assembly. In this paper, we report that forms liquid condensates appear to enable filament growth. Given transient nature these droplets, focus on properties vimentin-Y117L, has a point mutation leads formation but not IFs, enabling us study droplets detail. dissolve under 1,6-Hexanediol treatment decreasing concentration, confirming they are liquid, phase separated. These extensively wet actin stress fibers, rendering them resistant actin-binding drugs protecting from depolymerization. We show similar behavior occurs wild-type during its assembly into filaments.

Language: Английский

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Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells

The Journal of Cell Biology, Journal Year: 2024, Volume and Issue: 223(3)

Published: Jan. 19, 2024

Cytoplasmic dynein 1 (dynein) is the primary minus end–directed motor protein in most eukaryotic cells. Dynein remains an inactive conformation until formation of a tripartite complex comprising dynein, its regulator dynactin, and cargo adaptor. How this process activation occurs unclear since it entails three-protein inside crowded environs cell. Here, we employed live-cell, single-molecule imaging to visualize track fluorescently tagged dynein. First, observed that only ∼30% molecules bound microtubule (MT) engaged movement, too for short duration ∼0.6 s. Next, using high-resolution live fixed cells correlative light electron microscopy, discovered dynactin endosomal remained proximity each other MTs. We then two-color movement effected by single binding. Finally, performed long-term show movements are sufficient drive perinuclear region Taken together, search mechanism facilitated dynein’s frequent MT binding–unbinding kinetics: (i) futile event when does not encounter anchored MT, dissociates diffuses into cytoplasm, (ii) encounters upon binding, moves run. Several these runs undertaken succession long-range directed movement. In conclusion, demonstrate capture coupled step relies on reduction dimensionality enable transport cellulo behavior emerges from stochastic motor–cargo interactions.

Language: Английский

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MINFLUX reveals dynein stepping in live neurons

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(38)

Published: Sept. 10, 2024

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety cargoes, performing successive nanometer-sized steps within milliseconds. Due to limited spatiotemporal precision established methods tracking, current knowledge dynein stepping essentially slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization directly track CRISPR/Cas9-tagged endogenous with nanometer/millisecond living neurons. We show that primarily takes 8 nm steps, including frequent sideways but few backward steps. Strikingly, majority direction reversals between and anterograde movement occurred on time scale single (16 ms), suggesting rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or was unexpectedly rare. By analyzing dwell concluded rate-limiting process underlies mechanism, likely arising from just one adenosine 5′-triphosphate hydrolysis event being required each step. Our study underscores power elucidate changes underlying protein function cells.

Language: Английский

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Axonal transport of CHMP2b is regulated by kinesin-binding protein and disrupted by CHMP2b^intron5

Life Science Alliance, Journal Year: 2025, Volume and Issue: 8(5), P. e202402934 - e202402934

Published: Feb. 28, 2025

CHMP2b is a core component of the ESCRT pathway that catalyzes formation multivesicular bodies for endolysosomal protein degradation. Although mutation/loss-of-function promotes presynaptic dysfunction and degeneration, indicating its critical role in homeostasis, mechanisms responsible localization recruitment to synapses remain unclear. Here, we characterize axonal trafficking show transport boutons, as well cotransport with other proteins, are regulated by neuronal activity. In contrast, frontotemporal dementia–causative intron5 mutation exhibits little processive movement or presence absence Instead, vesicles exhibit oscillatory behavior reminiscent tug-of-war between kinesin dynein motor proteins. We this phenotype caused deficient binding kinesin-binding protein, which identify key regulator transport. These findings shed light on synaptic localization, their disruption .

Language: Английский

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Fat traffic control: S-acylation in axonal transport

Molecular Pharmacology, Journal Year: 2025, Volume and Issue: unknown, P. 100039 - 100039

Published: April 1, 2025

Language: Английский

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Cargo specificity, regulation, and therapeutic potential of cytoplasmic dynein

Experimental & Molecular Medicine, Journal Year: 2024, Volume and Issue: 56(4), P. 827 - 835

Published: April 1, 2024

Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, has a single isoform, which raises questions about cargo specificity and regulatory mechanisms. The precision of dynein-mediated is governed by multitude factors, including temperature, phosphorylation, microtubule track, interactions with family activating adaptor proteins. Activating adaptors are particular importance because they not only activate unidirectional motility but also connect diverse array cargoes motor. Therefore, it unsurprising that dysregulation dynein-activating machinery can lead to diseases such as spinal muscular atrophy, lower extremity, dominant. Here, we discuss within vitro, present several methodologies employed track motion We highlight newly identified their roles regulating dynein. Finally, explore potential therapeutic applications manipulating address linked malfunction.

Language: Английский

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MINFLUX Reveals Dynein Stepping in Live Neurons

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 23, 2024

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety cargoes, performing successive nanometer-sized steps within milliseconds. Due to limited spatiotemporal precision established methods tracking, current knowledge dynein stepping essentially slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization directly track CRISPR/Cas9-tagged endogenous with nanometer/millisecond living neurons. We show that primarily takes 8 nm steps, including frequent sideways but few backward steps. Strikingly, majority direction reversals between and anterograde movement occurred on time scale single (16 ms), suggesting rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or was unexpectedly rare. By analyzing dwell concluded rate-limiting process underlies mechanism whereby consumes one adenosine 5’-triphosphate (ATP) per step. Our study underscores power elucidate changes underlying protein function cells.

Language: Английский

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Episodic Transport of Protein Aggregates Achieves a Positive Size Selectivity in Aggresome Formation

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 7, 2024

Eukaryotic cells direct toxic misfolded proteins to various protein quality control pathways based on their chemical features and aggregation status. Aggregated are targeted selective autophagy or specifically sequestered into the "aggresome," a perinuclear inclusion at microtubule-organizing center (MTOC). However, mechanism for selectively sequestering aggregates aggresome remains unclear. To investigate formation, we reconstituted MTOC-directed aggregate transport in

Language: Английский

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Systems mapping of bidirectional endosomal transport through the crowded cell

Current Biology, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 1, 2024

Language: Английский

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CCSer2 gates dynein activity at the cell periphery

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 13, 2024

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from correct cellular region. How dynein’s trafficking activity regulated in or space remains poorly understood. Here, we identify CCSer2 as first known protein to gate dynein spatial dimension. promotes migration developing zebrafish primordium cells cultured human by facilitating are acted on cortically localized dynein. inhibits interaction its regulator Ndel1 exclusively cell periphery, resulting activation. Our findings suggest specificity achieved localization proteins disinhibit Ndel1. We propose defines broader class activate distinct microenvironments via inhibition.

Language: Английский

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