Precise control of microtubule disassembly in living cells

Microtubules (MTs) are components of the evolutionarily conserved cytoskeleton, which tightly regulates various cellular activities. Our understanding of MTs is largely based on MT-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific MT populations due to their slow effects on the entire pool of MTs in cells. To address this limitation, we have used chemogenetics and optogenetics to disassemble specific MT subtypes by rapid recruitment of engineered MT-cleaving enzymes. Acute MT disassembly swiftly halted vesicular trafficking and lysosome dynamics. We also used this approach to disassemble MTs specifically modified by tyrosination and several MT-based structures including primary cilia, mitotic spindles, and intercellular bridges. These effects were rapidly reversed by inhibiting the activity or MT association of the cleaving enzymes. The disassembly of targeted MTs with spatial and temporal accuracy enables to uncover new insights of how MTs precisely regulate cellular architectures and functions.

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Microtubule-based mitotic spindles contain a micron-sized mixed-nucleotide zone

10.1101/2021.07.23.453504 ◽

2021 ◽

Author(s):

Cedric Castrogiovanni ◽

Alessio V. Inchingolo ◽

Jonathan U. Harrison ◽

Damian Dudka ◽

Onur Sen ◽

...

Keyword(s):

Mitotic Spindle ◽

Computational Simulation ◽

Living Cells ◽

Mitotic Spindles ◽

In Cells

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores (K-fibres). Here we reveal that K-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled EBs, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro. In living cells HURP accumulates on the ends of depolymerising K-fibres, whilst avoiding recruitment to nascent polymerising K-fibres. This gives rise to a growing 'HURP-gap' which we can recapitulate in a minimal computational simulation. We therefore postulate that the K-fibre lattice contains a dynamic, micron-sized mixed-nucleotide zone.

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Therapeutically actionable signaling node to rescue AURKA driven loss of primary cilia in VHL-deficient cells

Scientific Reports ◽

10.1038/s41598-021-89933-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pratim Chowdhury ◽

Dimuthu Perera ◽

Reid T. Powell ◽

Tia Talley ◽

Durga Nand Tripathi ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Primary Cilia ◽

Mtor Inhibitor ◽

E3 Ubiquitin Ligase ◽

Xenograft Model ◽

Aurora Kinase ◽

Tumor Burden ◽

Aurora Kinase A ◽

Von Hippel Lindau ◽

In Cells

AbstractLoss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.

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Probing nucleus-enriched proteins in single living cells via subcellular-resolved plasmonic immunosandwich assay

The Analyst ◽

10.1039/d1an00003a ◽

2021 ◽

Author(s):

Jia Liu ◽

Dan Xie ◽

Zhen Liu

Keyword(s):

Living Cells ◽

Nuclear Proteins ◽

Biological Functions ◽

Abnormal Expression ◽

Single Living Cells ◽

Single Living ◽

In Cells

Nuclear proteins are crucial in cells and are greatly linked to various biological functions. Abnormal expression of nuclear proteins is associated with many diseases ranging from inflammation to cancer. However,...

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280: Primary Cilia and Fluid Flow Establish the Orientation of Mitotic Spindles

American Journal of Kidney Diseases ◽

10.1053/j.ajkd.2010.02.287 ◽

2010 ◽

Vol 55 (4) ◽

pp. B101

Author(s):

Neeraj Sharma ◽

Bradley K. Yoder

Keyword(s):

Fluid Flow ◽

Primary Cilia ◽

Mitotic Spindles

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Human Enhancer of Invasion-Cluster, a Coiled-Coil Protein Required for Passage through Mitosis

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3957-3971.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3957-3971 ◽

Cited By ~ 14

Author(s):

Margret B. Einarson ◽

Edna Cukierman ◽

Duane A. Compton ◽

Erica A. Golemis

Keyword(s):

Cell Cycle ◽

Coiled Coil ◽

Cell Cycle Checkpoints ◽

Mitotic Spindles ◽

Human Genes ◽

Hydroxyurea Treatment ◽

Evolutionarily Conserved ◽

Defects Analysis ◽

Spindle Function

ABSTRACT In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.

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polo, a mitotic mutant of Drosophila displaying abnormal spindle poles

Journal of Cell Science ◽

10.1242/jcs.89.1.25 ◽

1988 ◽

Vol 89 (1) ◽

pp. 25-38 ◽

Cited By ~ 5

Author(s):

C.E. Sunkel ◽

D.M. Glover

Keyword(s):

High Frequency ◽

Sex Chromosome ◽

Living Cells ◽

Male Meiosis ◽

Mitotic Spindles ◽

Spindle Poles ◽

Immunofluorescence Studies ◽

Meiotic Spindles ◽

Circular Arrangement ◽

Chromosome Disjunction

Neuroblast cells in larvae homozygous for mutant alleles of the locus polo show a high frequency of metaphases in which the chromosomes have a circular arrangement, and anaphase figures in which chromosomes appear to be randomly oriented with respect to at least one of the spindle poles. These defects appear to lead to the production of polyploid cells. Sex chromosome disjunction is affected in male meiosis, primarily in the second division, and the meiotic spindles of living cells are abnormal. One allele is a larval lethal, whereas another is semi-lethal with about 7% of homozygotes surviving as adults. Embryos from homozygous polo females have aberrant mitotic spindles that are highly branched and have broad poles. Immunofluorescence studies with an antibody that recognizes an antigen associated with the centrosome indicate that the organization of this organelle is disrupted in the mutant embryos.

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A FRET-based ratiometric two-photon fluorescent probe for superoxide anion detection and imaging in living cells and tissues

The Analyst ◽

10.1039/c8an02196a ◽

2019 ◽

Vol 144 (5) ◽

pp. 1704-1710 ◽

Cited By ~ 2

Author(s):

Shan Yao ◽

Chunli Ma ◽

Yanbing Lu ◽

Xianzhe Wei ◽

Xiaowen Feng ◽

...

Keyword(s):

Fluorescent Probe ◽

Superoxide Anion ◽

Living Cells ◽

Anion Detection ◽

Two Photon ◽

Dual Channels ◽

In Cells

A FRET-based ratiometric two-photon fluorescent probe for superoxide anion detection and imaging in cells and tissue was reported with dual-channels.

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Alp7-Mto1 and Alp14 synergize to promote interphase microtubule regrowth from the nuclear envelope

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz038 ◽

2019 ◽

Vol 11 (11) ◽

pp. 944-955 ◽

Cited By ~ 2

Author(s):

Wenyue Liu ◽

Fan Zheng ◽

Yucai Wang ◽

Chuanhai Fu

Keyword(s):

Nuclear Envelope ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Microtubule Assembly ◽

Dependent Manner ◽

Microtubule Nucleation ◽

Microtubule Organizing Centers ◽

Evolutionarily Conserved ◽

Microtubule Growth ◽

In Cells

Abstract Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers (MTOCs), including the nuclear envelope (NE) and pre-existing microtubules. The evolutionarily conserved proteins Mto1/CDK5RAP2 and Alp14/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation. However, it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs, particularly the NE, and how these proteins coordinate to organize microtubule assembly. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, efficient interphase microtubule growth from the NE requires Alp7/TACC, Alp14/TOG/XMAP215, and Mto1/CDK5RAP2. The absence of Alp7, Alp14, or Mto1 compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization. We further demonstrate that Alp7 and Mto1 interdependently localize to the NE in cells without microtubules and that Alp14 localizes to the NE in an Alp7 and Mto1-dependent manner. Tethering Mto1 to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE. Hence, our study delineates that Alp7, Alp14, and Mto1 work in concert to regulate interphase microtubule regrowth on the NE.

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Intrinsic Disorder-Based Emergence in Cellular Biology: Physiological and Pathological Liquid-Liquid Phase Transitions in Cells

Polymers ◽

10.3390/polym11060990 ◽

2019 ◽

Vol 11 (6) ◽

pp. 990 ◽

Cited By ~ 17

Author(s):

April L. Darling ◽

Boris Y. Zaslavsky ◽

Vladimir N. Uversky

Keyword(s):

Phase Transitions ◽

Phase Separation ◽

Liquid Phase ◽

Intrinsic Disorder ◽

Living Cells ◽

Cellular Biology ◽

Physiological Functions ◽

Pathological Conditions ◽

Liquid Liquid Phase Separation ◽

In Cells

The visible outcome of liquid-liquid phase transitions (LLPTs) in cells is the formation and disintegration of various proteinaceous membrane-less organelles (PMLOs). Although LLPTs and related PMLOs have been observed in living cells for over 200 years, the physiological functions of these transitions (also known as liquid-liquid phase separation, LLPS) are just starting to be understood. While unveiling the functionality of these transitions is important, they have come into light more recently due to the association of abnormal LLPTs with various pathological conditions. In fact, several maladies, such as various cancers, different neurodegenerative diseases, and cardiovascular diseases, are known to be associated with either aberrant LLPTs or some pathological transformations within the resultant PMLOs. Here, we will highlight both the physiological functions of cellular liquid-liquid phase transitions as well as the pathological consequences produced through both dysregulated biogenesis of PMLOs and the loss of their dynamics. We will also discuss the potential downstream toxic effects of proteins that are involved in pathological formations.

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