Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia

Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.

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Development for Dynamic Live Cell Imaging by Cryo-Electron Tomography and Stem

Biophysical Journal ◽

10.1016/j.bpj.2012.11.1962 ◽

2013 ◽

Vol 104 (2) ◽

pp. 354a

Author(s):

Atsuko H. Iwane ◽

Ruriko Ogawa ◽

Rina Nagai ◽

Akihiro Kawamoto ◽

Kazuhiro Aoyama

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Electron Tomography ◽

Live Cell ◽

Cryo Electron Tomography

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C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

10.1101/060855 ◽

2016 ◽

Cited By ~ 3

Author(s):

Stefanie Redemann ◽

Johannes Baumgart ◽

Norbert Lindow ◽

Sebastian Fürthauer ◽

Ehssan Nazockdast ◽

...

Keyword(s):

Light Microscopy ◽

Mitotic Spindle ◽

Live Cell Imaging ◽

Large Scale ◽

Cell Imaging ◽

Electron Tomography ◽

Mitotic Spindles ◽

C Elegans ◽

Microtubule Growth ◽

Segregation Of Chromosomes

AbstractThe mitotic spindle ensures the faithful segregation of chromosomes. To discover the nature of the crucial centrosome-to-chromosome connection during mitosis, we combined the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging. Using tomography, we reconstructed the positions of all microtubules in 3D, and identified their plus- and minus-ends. We classified them as kinetochore (KMTs), spindle (SMTs), or astral microtubules (AMTs) according to their positions, and quantified distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs are directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

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Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces

F1000Research ◽

10.12688/f1000research.23598.2 ◽

2020 ◽

Vol 9 ◽

pp. 449

Author(s):

Melanie B. Horev ◽

Yishaia Zabary ◽

Revital Zarka ◽

Simona Sorrentino ◽

Ohad Medalia ◽

...

Keyword(s):

Live Cell Imaging ◽

Platelet Adhesion ◽

Cell Imaging ◽

Electron Tomography ◽

Collagen Iv ◽

Short Term ◽

Functionalized Surfaces ◽

Cryo Electron Tomography ◽

Coated Surfaces ◽

Adhesion Process

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

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Balancing the length of distal tip is key for stability and signalling function of primary cilia

10.1101/2021.06.04.447095 ◽

2021 ◽

Author(s):

Taishi Kanamaru ◽

Annett Neuner ◽

Bahtiyar Kurtulmus ◽

Gislene Pereira

Keyword(s):

Sonic Hedgehog ◽

Live Cell Imaging ◽

Primary Cilia ◽

Cell Imaging ◽

Growth Inhibitor ◽

Distal Segment ◽

Segment Length ◽

Functional Transition ◽

Sensory Cilia ◽

Signalling Transduction

Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia are formed by middle and distal segments, in analogy to sensory cilia of lower eukaryotes. The analysis of middle/distal segmentation indicated that perturbations leading to cilia over-elongation influenced middle or distal segment length with a different impact on cilia behaviour. We identified Septins as novel repressors of distal segment growth. We show that Septins control the localisation of MKS3 and CEP290 required for a functional transition zone, and through this the entrance of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor, into the cilium. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that distal segment over-extension increased cilia excision events and decreased SHH activation. Our data underlies the importance of understanding cilia segmentation for length control and cilia-dependent signalling.

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Closed mitosis requires local disassembly of the nuclear envelope

10.1101/779769 ◽

2019 ◽

Cited By ~ 1

Author(s):

Gautam Dey ◽

Siân Culley ◽

Scott Curran ◽

Ricardo Henriques ◽

Wanda Kukulski ◽

...

Keyword(s):

Nuclear Envelope ◽

Live Cell Imaging ◽

Cell Imaging ◽

Electron Tomography ◽

Classical Model ◽

Nuclear Fission ◽

Nuclear Compartment ◽

Nuclear Compartments ◽

Intact Nucleus ◽

Narrow Bridge

At the end of mitosis, eukaryotic cells must segregate both copies of their replicated genome into two new nuclear compartments (1). They do this either by first dismantling and later reassembling the nuclear envelope in a so called “open mitosis”, or by reshaping an intact nucleus and then dividing into two in a “closed mitosis” (2, 3). However, while mitosis has been studied in a wide variety of eukaryotes for over a century (4), it is not known how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment (5). In studying this problem in the fission yeast Schizosaccharomyces pombe, a classical model for closed mitosis (5), we use genetics, live cell imaging and electron tomography to show that nuclear fission is achieved via local disassembly of the nuclear envelope (NE) within the narrow bridge that links segregating daughter nuclei. In doing so, we identify a novel inner NE-localised protein Les1 that restricts the process of local NE breakdown (local NEB) to the bridge midzone and prevents the leakage of material from daughter nuclei. The mechanics of local NEB in a closed mitosis closely mirror those of NEB in open mitosis (3), revealing an unexpectedly deep conservation of nuclear remodelling mechanisms across diverse eukaryotes.

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Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy

Traffic ◽

10.1111/tra.12557 ◽

2018 ◽

Vol 19 (5) ◽

pp. 354-369 ◽

Cited By ~ 29

Author(s):

Job Fermie ◽

Nalan Liv ◽

Corlinda ten Brink ◽

Elly G. van Donselaar ◽

Wally H. Müller ◽

...

Keyword(s):

Electron Microscopy ◽

Live Cell Imaging ◽

Cell Imaging ◽

3D Structure ◽

Live Cell ◽

3D Electron Microscopy ◽

3D Electron

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Ionic Liquid: Complexity in Structure and Dynamics, Interaction with Proteins and In Situ Generation of Metal Nano-clusters for Live Cell Imaging

Proceedings of the National Academy of Sciences India Section A Physical Sciences ◽

10.1007/s40010-018-0516-4 ◽

2018 ◽

Vol 88 (3) ◽

pp. 425-430 ◽

Cited By ~ 2

Author(s):

Somen Nandi ◽

Kankan Bhattacharyya

Keyword(s):

Ionic Liquid ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Structure And Dynamics ◽

In Situ Generation

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Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development

Nature Communications ◽

10.1038/s41467-021-20894-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

René Schneider ◽

Kris van’t Klooster ◽

Kelsey L. Picard ◽

Jasper van der Gucht ◽

Taku Demura ◽

...

Keyword(s):

Cell Walls ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Quantitative Microscopy ◽

Microtubule Severing ◽

Single Cell Imaging ◽

Spatio Temporal

AbstractPlants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

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