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Nature Nov 2022ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation. In turn, force and actin filament (F-actin) nucleotide state...
ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation. In turn, force and actin filament (F-actin) nucleotide state regulate actin dynamics by tuning F-actin's engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP-F-actin and ADP-P-F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP-F-actin and ADP-P-F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.
Topics: Actin Cytoskeleton; Actins; Adenosine Diphosphate; Cryoelectron Microscopy; Microfilament Proteins; Solvents; Machine Learning; Protein Conformation
PubMed: 36289330
DOI: 10.1038/s41586-022-05366-w -
Nature Communications Jun 2023We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with...
We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)-6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.
Topics: Humans; Actin-Related Protein 2-3 Complex; Actins; Cell Movement; Germ-Line Mutation; Cytokines
PubMed: 37349293
DOI: 10.1038/s41467-023-39272-0 -
Journal of Bacteriology Mar 2024Actin and actin-like proteins form filamentous polymers that carry out important cellular functions in all domains of life. In this review, we sketch a map of the... (Review)
Review
Actin and actin-like proteins form filamentous polymers that carry out important cellular functions in all domains of life. In this review, we sketch a map of the function and regulation of actin-like proteins across bacteria, archaea, and eukarya, marking some of the that remain in this landscape. We focus particular attention on archaea because mapping the structure and function of cytoskeletal systems across this domain promises to help us understand the evolutionary relationship between the (mostly) mono-functional actin-like filaments found in bacteria and the multi-functional actin cytoskeletons that characterize eukaryotic cells.
Topics: Actins; Archaea; Cytoskeleton; Bacteria; Biological Evolution
PubMed: 38391233
DOI: 10.1128/jb.00348-23 -
Experimental & Molecular Medicine Jun 2023The WAVE regulatory complex (WRC), composed of five components-Cyfip1/Sra1, WAVE/Scar, Abi, Nap1/Nckap1, and Brk1/HSPC300-is essential for proper actin cytoskeletal... (Review)
Review
The WAVE regulatory complex (WRC), composed of five components-Cyfip1/Sra1, WAVE/Scar, Abi, Nap1/Nckap1, and Brk1/HSPC300-is essential for proper actin cytoskeletal dynamics and remodeling in eukaryotic cells, likely by matching various patterned signals to Arp2/3-mediated actin nucleation. Accumulating evidence from recent studies has revealed diverse functions of the WRC in neurons, demonstrating its crucial role in dictating the assembly of molecular complexes for the patterning of various trans-synaptic signals. In this review, we discuss recent exciting findings on the physiological role of the WRC in regulating synaptic properties and highlight the involvement of WRC dysfunction in various brain disorders.
Topics: Actins; Wiskott-Aldrich Syndrome Protein Family; Carrier Proteins; Neurons
PubMed: 37258575
DOI: 10.1038/s12276-023-01004-1 -
Proceedings of the National Academy of... Sep 2022Gene expression is tightly regulated by RNA-binding proteins (RBPs) to facilitate cell survival, differentiation, and migration. Previous reports have shown the...
Gene expression is tightly regulated by RNA-binding proteins (RBPs) to facilitate cell survival, differentiation, and migration. Previous reports have shown the importance of the Insulin-like Growth Factor II mRNA-Binding Protein (IGF2BP1/IMP1/ZBP1) in regulating RNA fate, including localization, transport, and translation. Here, we generated and characterized a knockout mouse to study RBP regulation. We report that IGF2BP1 is essential for proper brain development and neonatal survival. Specifically, these mice display disorganization in the developing neocortex, and further investigation revealed a loss of cortical marginal cell density at E17.5. We also investigated migratory cell populations in the IGF2BP1[Formula: see text] mice, using BrdU labeling, and detected fewer mitotically active cells in the cortical plate. Since RNA localization is important for cellular migration and directionality, we investigated the regulation of -actin messenger RNA (mRNA), a well-characterized target with established roles in cell motility and development. To aid in our understanding of RBP and target mRNA regulation, we generated mice with endogenously labeled -actin mRNA (IGF2BP1[Formula: see text]; -actin-MS2[Formula: see text]). Using endogenously labeled -actin transcripts, we report IGF2BP1[Formula: see text] neurons have increased transcription rates and total -actin protein content. In addition, we found decreased transport and anchoring in knockout neurons. Overall, we present an important model for understanding RBP regulation of target mRNA.
Topics: Actins; Animals; Brain; Cell Movement; Mice; Mice, Knockout; Neurons; RNA, Messenger; RNA-Binding Proteins
PubMed: 36067310
DOI: 10.1073/pnas.2208465119 -
The FEBS Journal Dec 2022When the plasma membrane (PM) detaches from the underlying actin cortex, the PM expands according to intracellular pressure and a spherical membrane protrusion called a... (Review)
Review
When the plasma membrane (PM) detaches from the underlying actin cortex, the PM expands according to intracellular pressure and a spherical membrane protrusion called a bleb is formed. This bleb retracts when the actin cortex is reassembled underneath the PM. Whereas this phenomenon seems simple at first glance, there are many interesting, unresolved cell biological questions in each process. For example, what is the membrane source to enlarge the surface area of the PM during rapid bleb expansion? What signals induce actin reassembly for bleb retraction, and how is cytoplasmic fluidity regulated to allow rapid membrane deformation during bleb expansion? Furthermore, emerging evidence indicates that cancer cells use blebs for invasion, but little is known about how molecules that are involved in bleb formation, expansion, and retraction are coordinated for directional amoeboid migration. In this review, we discuss the molecular mechanisms involved in the regulation of blebs, which have been revealed by various experimental systems.
Topics: Humans; Actins; Calcium; Cell Membrane; Cytoplasm; Cytoskeleton
PubMed: 34614290
DOI: 10.1111/febs.16220 -
Journal of Molecular Cell Biology Aug 2023Primary cilia are microtubule-based cell organelles important for cellular communication. Since they are involved in the regulation of numerous signalling pathways,...
Primary cilia are microtubule-based cell organelles important for cellular communication. Since they are involved in the regulation of numerous signalling pathways, defects in cilia development or function are associated with genetic disorders, collectively called ciliopathies. Besides their ciliary functions, recent research has shown that several ciliary proteins are involved in the coordination of the actin cytoskeleton. Although ciliary and actin phenotypes are related, the exact nature of their interconnection remains incompletely understood. Here, we show that the protein BBS6, associated with the ciliopathy Bardet-Biedl syndrome, cooperates with the actin-bundling protein Fascin-1 in regulating filopodia and ciliary signalling. We found that loss of Bbs6 affects filopodia length potentially via attenuated interaction with Fascin-1. Conversely, loss of Fascin-1 leads to a ciliary phenotype, subsequently affecting ciliary Wnt signalling, possibly in collaboration with BBS6. Our data shed light on how ciliary proteins are involved in actin regulations and provide new insight into the involvement of the actin regulator Fascin-1 in ciliogenesis and cilia-associated signalling. Advancing our knowledge of the complex regulations between primary cilia and actin dynamics is important to understand the pathogenic consequences of ciliopathies.
Topics: Humans; Actins; Ciliopathies; Wnt Signaling Pathway
PubMed: 37015875
DOI: 10.1093/jmcb/mjad022 -
Communications Biology Sep 2021The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the...
The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.
Topics: Actin Cytoskeleton; Actins; Carrier Proteins; Humans; Microfilament Proteins
PubMed: 34584211
DOI: 10.1038/s42003-021-02653-6 -
Journal of Biomolecular Structure &... May 2023Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer,...
Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2) - which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin.Communicated by Ramaswamy H. Sarma.
Topics: Actins; Formins; Microfilament Proteins; Actin Cytoskeleton
PubMed: 35343388
DOI: 10.1080/07391102.2022.2050947 -
The EMBO Journal May 2023Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides...
Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.
Topics: Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Cytoskeleton; Actins
PubMed: 36939020
DOI: 10.15252/embj.2022113008