CryoET shows cofilactin filaments inside the microtubule lumen DOI Creative Commons
Ventura Santos C, Stephen L. Rogers, Andrew P. Carter

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: March 31, 2023

Abstract Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) Drosophila S2 cell protrusions found the microtubule lumen, which resemble those reported recently in human HAP1 cells. frequency increased upon inhibition sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) with small-molecule drug thapsigargin. Subtomogram averaging showed luminal adopt a helical structure reminiscent cofilin-bound actin (cofilactin). Consistent this, cofilin was activated cells under same conditions filament occurrence. Furthermore, RNAi knock-down reduced cofilactin morphology. These results suggest activation stimulates its on

Language: Английский

Fascin structural plasticity mediates flexible actin bundle construction DOI Creative Commons
Rui Gong, Matthew J. Reynolds, Keith R. Carney

et al.

Nature Structural & Molecular Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 20, 2025

Abstract Fascin cross-links actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia and stereocilia. dysregulation drives aberrant cell migration during metastasis, fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-EM, cryo-electron tomography coupled with custom denoising computational modeling to probe human fascin-1’s F-actin cross-linking mechanisms across spatial scales. Our cross-bridge structure reveals an asymmetric binding conformation is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements, variability analysis simulations show how structural plasticity enables bridge varied interfilament orientations, accommodating mismatches between F-actin’s helical symmetry packing. Tomography many-filament uncover geometric rules underlying emergent patterns, well accumulation unfavorable limit size. Collectively, this work shows harnesses fine-tuned nanoscale dynamics build regulate micron-scale bundles.

Language: Английский

Citations

2
Filopodia In Vitro and In Vivo DOI Creative Commons
Thomas C. A. Blake, Jennifer L. Gallop

Annual Review of Cell and Developmental Biology, Journal Year: 2023, Volume and Issue: 39(1), P. 307 - 329

Published: July 5, 2023

Filopodia are dynamic cell surface protrusions used for motility, pathogen infection, and tissue development. The molecular mechanisms determining how where filopodia grow retract need to integrate mechanical forces membrane curvature with extracellular signaling the broader state of cytoskeleton. involved actin regulatory machinery nucleates, elongates, bundles filaments separately from underlying cortex. refined geometry filopodia, importance context, high spatiotemporal resolution required, degree redundancy all limit current models. New technologies improving opportunities functional insight, reconstitution in vitro purified components, endogenous genetic modification, inducible perturbation systems, study multicellular environments. In this review, we explore recent advances conceptual models form, molecules process, our latest understanding vivo.

Language: Английский

Citations

25
Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules DOI Creative Commons
Saikat Chakraborty, Antonio Martínez-Sánchez, Florian Beck

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(5)

Published: Jan. 31, 2025

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most which are known to bind MT outer surface. However, electron microscopy (EM) has long ago revealed presence particles inside lumens MTs, yet unknown identity and function. Here, we use cryogenic tomography (cryo-ET) analyze three-dimensional (3D) organization structures lumenal in primary hippocampal neurons, human induced pluripotent stem cell-derived differentiated P19 cells. We obtain situ density maps several from respective cells detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with modeling suggest that a subset could be tubulin-binding cofactors (TBCs) bound tubulin monomers. A different smaller particles, remains unidentified, exhibits densities bridge across protofilaments. show increased particle concentration within MTs concomitant differentiation correlates higher curvatures. Enrichment around lattice defects at freshly polymerized open-ends suggests protective role. Together identified resemblance TBCs, these results hint role local proteostasis for maintenance MTs.

Language: Английский

Citations

1
Doublecortin restricts neuronal branching by regulating tubulin polyglutamylation DOI Creative Commons
Muriel Sébastien, Alexandra Paquette, Emily N. P. Prowse

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Feb. 18, 2025

Doublecortin is a neuronal microtubule-associated protein that regulates microtubule structure in neurons. Mutations cause lissencephaly and subcortical band heterotopia by impairing migration. We use CRISPR/Cas9 to knock-out the gene induced pluripotent stem cells differentiate into cortical DCX-KO neurons show reduced velocities of nuclear movements an increased number neurites early development, consistent with previous findings. Neurite branching regulated host proteins, as well polymerization dynamics. However, EB comet dynamics are unchanged Rather, we observe significant reduction α-tubulin polyglutamylation Polyglutamylation levels rescued expression or TTLL11, glutamylase. Using U2OS orthogonal model system, DCX TTLL11 act synergistically promote polyglutamylation. propose acts positive regulator restricts neurite branching. Our results indicate unexpected role for homeostasis tubulin code. Lissencephaly severe neurodevelopmental disease often caused mutations Dcx gene. human cellular lissencephaly, authors report activating

Language: Английский

Citations

1
A kinesin-1 variant reveals motor-induced microtubule damage in cells DOI Creative Commons
Breane Budaitis, Somayesadat Badieyan,

Yang Yue

et al.

Current Biology, Journal Year: 2022, Volume and Issue: 32(11), P. 2416 - 2429.e6

Published: May 2, 2022

Language: Английский

Citations

32
Cellular cartography: Towards an atlas of the neuronal microtubule cytoskeleton DOI Creative Commons
Malina K. Iwanski, Lukas C. Kapitein

Frontiers in Cell and Developmental Biology, Journal Year: 2023, Volume and Issue: 11

Published: March 22, 2023

Microtubules, one of the major components cytoskeleton, play a crucial role during many aspects neuronal development and function, such as polarization axon outgrowth. Consequently, microtubule cytoskeleton has been implicated in neurodevelopmental neurodegenerative disorders. The polar nature microtubules is quintessential for their allowing them to serve tracks long-distance, directed intracellular transport by kinesin dynein motors. Most these motors move exclusively towards either plus- or minus-end some have shown preference dynamic stable microtubules, those bearing particular post-translational modification decorated specific microtubule-associated protein. Thus, it becomes important consider interplay features combinatorial effects on transport, well how different types are organized cell. Here, we discuss subsets terms tubulin isotypes, modifications, proteins, stability dynamicity, orientation. We highlight techniques used study and, using information from studies, try define composition, role, organization neurons.

Language: Английский

Citations

20
Ciliary tip actin dynamics regulate photoreceptor outer segment integrity DOI Creative Commons
Roly Megaw, Abigail R. Moye, Zhixian Zhang

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: May 21, 2024

Abstract As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs shed and remade daily. Defects in this process, due mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, mechanism photoreceptor disc generation is poorly understood. Here, we show deformation required genesis driven dynamic changes akin ectocytosis. We RPGR, leading RP gene, regulates actin-binding protein activity central process. Actin dynamics, formation, perturbed Rpgr mouse models, aborted shedding as ectosome-like vesicles, death visual loss. manipulation partially rescues this, suggesting pathway could be targeted therapeutically. These findings help define how actin-mediated control segment turnover.

Language: Английский

Citations

7
Doublecortin reinforces microtubules to promote growth cone advance in soft environments DOI Open Access
Alessandro Dema, Rabab A. Charafeddine, Jeffrey van Haren

et al.

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

Published: Feb. 29, 2024

Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, molecular function of DCX neuron morphogenesis remains unknown and controversial. We demonstrate that intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i

Language: Английский

Citations

6
Causes, costs and consequences of kinesin motors communicating through the microtubule lattice DOI Creative Commons
Kristen J. Verhey, Ryoma Ohi

Journal of Cell Science, Journal Year: 2023, Volume and Issue: 136(5)

Published: March 1, 2023

ABSTRACT Microtubules are critical for a variety of important functions in eukaryotic cells. During intracellular trafficking, molecular motor proteins the kinesin superfamily drive transport cellular cargoes by stepping processively along microtubule surface. Traditionally, has been viewed as simply track motility. New work is challenging this classic view showing that kinesin-1 and kinesin-4 can induce conformational changes tubulin subunits while they stepping. These appear to propagate such kinesins allosterically through lattice influence other on same track. Thus, plastic medium which motors microtubule-associated (MAPs) communicate. Furthermore, damage lattice. Damage be repaired incorporation new subunits, but too much leads breakage disassembly. addition loss not restricted ends filament rather, itself undergoes continuous repair remodeling. This understanding how their tracks engage allosteric interactions normal cell physiology.

Language: Английский

Citations

15
In situ studies of membrane biology by cryo-electron tomography DOI Creative Commons

Jenny Keller,

Rubén Fernández‐Busnadiego

Current Opinion in Cell Biology, Journal Year: 2024, Volume and Issue: 88, P. 102363 - 102363

Published: April 26, 2024

Cryo-electron tomography (cryo-ET) allows high resolution 3D imaging of biological samples in near-native environments. Thus, cryo-ET has become the method choice to analyze unperturbed organization cellular membranes. Here, we briefly discuss current workflows and their application study membrane biology situ, under basal pathological conditions.

Language: Английский

Citations

5