Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

Pineau, Judith; Pinon, Léa; Mesdjian, Olivier; Fattaccioli, Jacques; Lennon-Duménil, Ana-María; Pierobon, Paolo

doi:10.7554/elife.78330

Cited by 13 publications

(11 citation statements)

References 62 publications

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“…They also suggested the inhibitory effect of microtubules on actin network remodeling and membrane ruffling. A similar regulatory effect of microtubules has been reported by Pineau et al (2022) [ 33 ]. They demonstrated that microtubules and the GEF–H1–RhoA axis had an inhibitory effect on actin polymerization at the lymphocyte–antigen contact site.…”

Section: Discussionsupporting

confidence: 86%

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

Seta

Kawakatsu

Degawa

et al. 2023

IJMS

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Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

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Section: Discussionsupporting

confidence: 86%

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

Seta

Kawakatsu

Degawa

et al. 2023

IJMS

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“…In the course of optimizing the classification, an additional phenotype termed “patchy” was discovered. A similar distribution of multiple F‐actin polymerization spots was described at the cortex of T cells during immune synapse formation upon microtubule depolymerization 18 . The patchy fraction was affected by treatments with CS and NSC, indicating that it constitutes a bona fide biological phenotype to be further investigated.…”

Section: Discussionsupporting

confidence: 55%

Analysis of cortical cell polarity by imaging flow cytometry

Jespersen,

Harazin,

Bohn

et al. 2023

J of Cellular Biochemistry

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Metastasis is the main cause of cancer‐related death and therapies specifically targeting metastasis are highly needed. Cortical cell polarity (CCP) is a prometastatic property of circulating tumor cells affecting their ability to exit blood vessels and form new metastases that constitute a promising point of attack to prevent metastasis. However, conventional fluorescence microscopy on single cells and manual quantification of CCP are time‐consuming and unsuitable for screening regulators. In this study, we developed an imaging flow cytometry‐based method for high‐throughput screening of factors affecting CCP in melanoma cells. The artificial intelligence‐supported analysis method we developed is highly reproducible, accurate, and orders of magnitude faster than manual quantification. Additionally, this method is flexible and can be adapted to include additional cellular parameters. In a small‐scale pilot experiment using polarity‐, cytoskeleton‐, or membrane‐affecting drugs, we demonstrate that our workflow provides a straightforward and efficient approach for screening factors affecting CCP in cells in suspension and provide insights into the specific function of these drugs in this cellular system. The method and workflow presented here will facilitate large‐scale studies to reveal novel cell‐intrinsic as well as systemic factors controlling CCP during metastasis.

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“…In our experiments, LatA did not induce the formation of the static protrusive structure when the microtubule cytoskeleton was depolymerized priorly, an observation in line with Obino et al ( 2016 ). Microtubule‐mediated polarity cues for protrusion formation could involve regulating the phosphoinositide content of the plasma membrane (Golub & Caroni, 2005 ; Sugiyama et al , 2015 ), asymmetrically activating Rho signaling (Pineau et al , 2022 ) or providing direct interaction with membrane‐bending factors (Kelley et al , 2015 ). We think that, once these factors, likely to belong to the BAR protein superfamily, stably bind and bend membranes in the absence of actin cytoskeleton, subsequent microtubule depolymerization is no longer effective.…”

Section: Discussionmentioning

confidence: 99%

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters

et al. 2023

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B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces.Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cellsurface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membranelocalized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridgenetwork organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

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Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

Cited by 13 publications

References 62 publications

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

Analysis of cortical cell polarity by imaging flow cytometry

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters

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