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      Time-resolved ultrastructure of the cortical actin cytoskeleton in dynamic membrane blebs

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          Abstract

          Chikina et al. show preferential initiation of blebbing at local cytoskeleton weaknesses at filopodial bases when blebbing is induced by Arp2/3 complex inhibition. They also use correlative platinum replica electron microscopy to characterize actin cytoskeleton architecture in blebs at different stages of their expansion–retraction cycle.

          Abstract

          Membrane blebbing accompanies various cellular processes, including cytokinesis, apoptosis, and cell migration, especially invasive migration of cancer cells. Blebs are extruded by intracellular pressure and are initially cytoskeleton-free, but they subsequently assemble the cytoskeleton, which can drive bleb retraction. Despite increasing appreciation of physiological significance of blebbing, the molecular and, especially, structural mechanisms controlling bleb dynamics are incompletely understood. We induced membrane blebbing in human HT1080 fibrosarcoma cells by inhibiting the Arp2/3 complex. Using correlative platinum replica electron microscopy, we characterize cytoskeletal architecture of the actin cortex in cells during initiation of blebbing and in blebs at different stages of their expansion–retraction cycle. The transition to blebbing in these conditions occurred through an intermediate filopodial stage, whereas bleb initiation was biased toward filopodial bases, where the cytoskeleton exhibited local weaknesses. Different stages of the bleb life cycle (expansion, pausing, and retraction) are characterized by specific features of cytoskeleton organization that provide implications about mechanisms of cytoskeleton assembly and bleb retraction.

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          The Actin Cytoskeleton and Actin-Based Motility

          The actin cytoskeleton—a collection of actin filaments with their accessory and regulatory proteins—is the primary force-generating machinery in the cell. It can produce pushing (protrusive) forces through coordinated polymerization of multiple actin filaments or pulling (contractile) forces through sliding actin filaments along bipolar filaments of myosin II. Both force types are particularly important for whole-cell migration, but they also define and change the cell shape and mechanical properties of the cell surface, drive the intracellular motility and morphogenesis of membrane organelles, and allow cells to form adhesions with each other and with the extracellular matrix.
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            Characterization of two classes of small molecule inhibitors of Arp2/3 complex

            Polymerization of actin filaments directed by the Arp2/3 complex supports many types of cellular movements1. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones owing to the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-636 binds between Arp2 and Arp3 where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying Arp2/3 complex in living cells.
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              Non-equilibration of hydrostatic pressure in blebbing cells.

              Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry. In plants and fungi, protrusion is driven primarily by hydrostatic pressure. For hydrostatic pressure to drive localized protrusion in animal cells, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum or viscous liquid require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of approximately 10 microm and 10 s. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogeneous transients in the pressure field, and can be described in the framework of poroelasticity.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                jcb
                The Journal of Cell Biology
                Rockefeller University Press
                0021-9525
                1540-8140
                04 February 2019
                : 218
                : 2
                : 445-454
                Affiliations
                [1 ]Laboratory of Mechanisms of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia
                [2 ]Department of Biology, University of Pennsylvania, Philadelphia, PA
                Author notes
                Correspondence to Antonina Y. Alexandrova: tonya_alex@ 123456yahoo.com
                Tatyana M. Svitkina: svitkina@ 123456sas.upenn.edu

                A.S. Chikina’s present address is Cell Migration and Invasion and Spatio-temporal Regulation of Antigen Presentation teams, UMR144/U932 Institut Curie, Paris, France.

                Author information
                http://orcid.org/0000-0002-6801-7031
                http://orcid.org/0000-0002-8424-447X
                http://orcid.org/0000-0001-5071-2290
                Article
                201806075
                10.1083/jcb.201806075
                6363452
                30541746
                1c6d6845-cc7a-4827-b865-4bdb64bb03b0
                © 2019 Chikina et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

                History
                : 12 June 2018
                : 14 October 2018
                : 26 November 2018
                Funding
                Funded by: Russian Science Foundation, DOI https://doi.org/10.13039/501100006769;
                Award ID: 16-15-10288
                Funded by: National Institutes of Health, DOI https://doi.org/10.13039/100000002;
                Award ID: R01 GM095977
                Categories
                Research Articles
                Report
                37
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                Cell biology
                Cell biology

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