Lattice defects induce microtubule self-renewal

Schaedel, Laura; Triclin, Sarah; Chrétien, Denis; Abrieu, Ariane; Aumeier, Charlotte; Gaillard, Jérémie; Blanchoin, Laurent; Théry, Manuel; John, Karin

doi:10.1038/s41567-019-0542-4

Cited by 92 publications

(74 citation statements)

References 47 publications

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“…Fig. 1) or by kinesin-mediated disruption of the lattice 9,23,24 . Put another way, F-actin incorporation may occur in response to mechanical or structural stress 25 .…”

Section: Discussionmentioning

confidence: 99%

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

Paul

Mantell

Borucu

et al. 2019

Preprint

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Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organisation to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryoelectron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small-molecule induced, microtubule-based cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in-situ study of microtubule structure and contents.

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“…Fig. 1) or by kinesin-mediated disruption of the lattice 9,23,24 . Put another way, F-actin incorporation may occur in response to mechanical or structural stress 25 .…”

Section: Discussionmentioning

confidence: 99%

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

Paul

Mantell

Borucu

et al. 2019

Preprint

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“…We started by comparing the micropatterning efficiency of two proteins broadly used for micropatterning, NeutrAvidin 7,8,17 and Fibrinogen 11,18,19 . Except for figure 4, all micropatterns in this study were obtained following the LIMAP protocol 16 using a UV-projector in a fluorescence microscope and the photosensitizer 4-benzoylbenzyl)trimethylammonium bromide (BBTB), see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This led to breakthroughs in biology, allowing researchers, for example, to print cell adhesion proteins to constrain cell shape and thus reveal the physical basis of cell polarity 4 and spindle orientation during mitosis in cultured cells 5 . Concomitantly, the ability to print purified proteins onto glass brought better control in in vitro reconstitution studies, which strengthened our understanding of the dynamics of cytoskeleton contractility 6 , as well as the physiology of cytoskeletal polymers 7,8 . Finally, it was recently demonstrated that electron microscopy grids could be micropatterned with cell adhesion molecules 9,10 .…”

Section: Introductionmentioning

confidence: 99%

Fibrinogen anchors for micropatterning of active proteins and subcellular receptor relocalisation

Watson

Aich

Salvia

et al. 2020

Preprint

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Protein micropatterning is the process by which molecules are precisely deposited onto a substrate of choice. Over the past few decades, this technology has allowed numerous breakthroughs in biological sciences due to its extensive use in cell biology and in in vitro reconstitution. However, a major drawback in current technology is that micropatterning efficiency is highly variable between proteins, and proteins tend to lose activity on the pattern, which is particularly problematic when performing multiplexed micropatterning. Here, we describe a general method to enable micropatterning of virtually any protein at a high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, Fibrinogen, which we functionalized to bind to protein of interest or common purification tags (biotin, streptavidin, GFP). This allows high efficiency, homogeneous patterning of virtually any protein of interest. This method also protects proteins of interest from the potentially damaging patterning process, by binding them to the pattern after the patterning process. We show that this method enhances micropatterning on various substrates, facilitates multiplexed micropatterning by homogenizing the micropatterning efficiency of successive proteins, and enhances the on-pattern activity of fragile proteins like molecular motors. Furthermore, it allows the micropatterning of cells that could not be micropatterned previously due to poor patterning of their extracellular matrix. Last, we demonstrate that this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology, in particular to elucidate the molecular mechanisms underlying cross-talks between signalling cascades, symmetry breaking of the cell cortex or structural studies of membrane receptor in situ by Cryo-Electron Microscopy.

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“…These and other studies have led to the growing appreciation that lattice dynamics of even stable microtubules can provide access to luminal residues along the shaft of microtubule polymers. Recently, Schaedel et al, reported a passive breathing mechanism for microtubules in which α/β tubulin dimers can be evicted and replaced from the microtubule shaft via an energy consuming process 16 . SWI/SNF complexes are known to evict histones and other proteins from nucleosomes during chromatin remodeling [17][18][19] , suggesting the intriguing hypothesis that PBAF, and perhaps other SWI/SNF complexes, could be playing a similar role in evicting tubulin and/or other microtubule associated proteins during microtubule remodeling.…”

Section: Discussionmentioning

confidence: 99%

“…The pellet was removed and supernatants collected as whole cell extract for immunoblotting and immunoprecipitation analysis for soluble proteins. For subcellular fractionation, the pellet was collected after centrifugation as above, and resuspended in a hypotonic buffer (10 mM HEPES, pH 7.2, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 20 mM NaF, and 100 μM Na3VO4), and disrupted using a manual homogenizer for [15][16][17][18][19][20] times. Disruption of the cells was confirmed using a hemacytometer.…”

Section: Whole Cell Lysis and Sub-cellular Fractionationmentioning

confidence: 99%

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

Karki

Jangid

Seervai

et al. 2020

Preprint

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The chromatin modifier SETD2 was recently shown to be a dual-function methyltransferase that "writes" methyl marks on both chromatin and the mitotic spindle, revealing α-tubulin methylation as a new posttranslational modification of microtubules.Here, we report the first cytoskeletal "reader" for this SETD2 methyl mark: the polybromo protein PBRM1. We found PBRM1 directly binds the α-Tub-K40me3 mark on tubulin, and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 can assemble a PBAF complex in the absence of chromatin as revealed by mass spectrometry, and can recruit other PBAF complex components including SMARCA4 and ARID2 to α-tubulin.In addition to PBRM1, other PBAF components were also localized to the mitotic spindle and spindle pole. This PBAF localization was dependent on recruitment to microtubules by PBRM1, and loss of spindle-associated PBRM1/PBAF led to genomic instability as assessed by increased formation of micronuclei. These data reveal a previously unknown function for PBRM1 beyond its role remodeling chromatin, and expand the repertoire of chromatin remodelers involved in writing and reading methyl marks on the cytoskeleton.The results of this study lay the foundation for a new paradigm for the epigenetic machinery as chromatocytoskeletal modifiers, with coordinated nuclear and cytoskeletal functions.3 Introduction:

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Lattice defects induce microtubule self-renewal

Cited by 92 publications

References 47 publications

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

In situ cryo-electron tomography reveals filamentous actin within the microtubule lumen

Fibrinogen anchors for micropatterning of active proteins and subcellular receptor relocalisation

A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules

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