ABSTRACT
Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis.
Subject(s)
Actin Cytoskeleton/metabolism , Cofilin 1/metabolism , Destrin/metabolism , Actin-Related Protein 3/antagonists & inhibitors , Actin-Related Protein 3/genetics , Actin-Related Protein 3/metabolism , Animals , Cadherins/metabolism , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Survival , Cells, Cultured , Cofilin 1/antagonists & inhibitors , Cofilin 1/genetics , Destrin/deficiency , Destrin/genetics , Focal Adhesions/metabolism , Formins , Keratinocytes/cytology , Keratinocytes/metabolism , Mice , Mice, Knockout , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , NADPH Dehydrogenase/antagonists & inhibitors , NADPH Dehydrogenase/genetics , NADPH Dehydrogenase/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Skin/metabolism , Skin/pathologyABSTRACT
The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.
Subject(s)
Actin Cytoskeleton/metabolism , Actin-Related Protein 2/physiology , Actin-Related Protein 3/physiology , Axons/physiology , Cell Differentiation/physiology , Embryonic Stem Cells/physiology , Growth Cones/physiology , Pseudopodia/metabolism , Actin Cytoskeleton/physiology , Actin-Related Protein 2/antagonists & inhibitors , Actin-Related Protein 3/antagonists & inhibitors , Animals , Chick Embryo , Chickens , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/physiology , Primary Cell Culture , Pseudopodia/physiology , Sensory Receptor Cells/cytology , Sensory Receptor Cells/physiologyABSTRACT
Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. 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; this is because of 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 the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 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 the Arp2/3 complex in living cells.
