ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

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Rho Family Gtpase Cdc42 Is Essential for the Actin-Based Motility of Shigella in Mammalian Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.11.1905 ◽

1999 ◽

Vol 191 (11) ◽

pp. 1905-1920 ◽

Cited By ~ 46

Author(s):

Toshihiko Suzuki ◽

Hitomi Mimuro ◽

Hiroaki Miki ◽

Tadaomi Takenawa ◽

Takuya Sasaki ◽

...

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Host Cells ◽

Actin Dynamics ◽

Cloud Formation ◽

Actin Assembly ◽

Egg Extracts ◽

Rho Family ◽

Green Fluorescent

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP–depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP–actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein–tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG–N-WASP–Arp2/3 complex formed on intracellular Shigella.

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A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e04-09-0774 ◽

2005 ◽

Vol 16 (2) ◽

pp. 964-975 ◽

Cited By ~ 303

Author(s):

Defne Yarar ◽

Clare M. Waterman-Storer ◽

Sandra L. Schmid

Keyword(s):

Cell Surface ◽

Mammalian Cells ◽

Actin Polymerization ◽

Cell Surface Receptor ◽

Actin Dynamics ◽

Coated Vesicle ◽

Vesicle Formation ◽

Actin Assembly ◽

Electron Microscopic ◽

Surface Receptor

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

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Side-binding proteins modulate actin filament dynamics

10.1101/008128 ◽

2014 ◽

Author(s):

Alvaro H. Crevenna ◽

Marcelino Arciniega ◽

Aurelie Dupont ◽

Kaja Kowalska ◽

Oliver Lange ◽

...

Keyword(s):

Actin Filament ◽

Brownian Dynamics ◽

Actin Polymerization ◽

Actin Dynamics ◽

Central Feature ◽

Filament Dynamics ◽

Filament Structure ◽

The Rich ◽

Dynamics Simulations ◽

Pointed End

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of the filament to polymerize and depolymerize at its ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. Here, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by proteins that bind to the lateral filament surface. We also show that the less dynamic end, called the pointed-end, has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of filament flexibility and Brownian dynamics simulations suggest that the observed kinetic diversity arises from structural alteration. Tuning filament kinetics by exploiting the natural malleability of the actin filament structure may be a ubiquitous mechanism to generate the rich variety of observed cellular actin dynamics.

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Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0383 ◽

2012 ◽

Vol 23 (15) ◽

pp. 2891-2904 ◽

Cited By ~ 57

Author(s):

Jackie Cheng ◽

Alexandre Grassart ◽

David G. Drubin

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Type I ◽

Actin Assembly ◽

Significant Delay ◽

Src Homology 3 ◽

Integral Role ◽

Src Homology 3 Domain ◽

Src Homology ◽

Endocytic Vesicles

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.

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Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009167117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19904-19913 ◽

Cited By ~ 2

Author(s):

Caner Akıl ◽

Linh T. Tran ◽

Magali Orhant-Prioux ◽

Yohendran Baskaran ◽

Edward Manser ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Calcium Binding ◽

Mammalian Cells ◽

Actin Polymerization ◽

Calcium Release ◽

Duplication Event ◽

Cellular Level ◽

Actin Dynamics

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

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Negative Regulation of Yeast Eps15-like Arp2/3 Complex Activator, Pan1p, by the Hip1R-related Protein, Sla2p, during Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0788 ◽

2007 ◽

Vol 18 (2) ◽

pp. 658-668 ◽

Cited By ~ 31

Author(s):

Jiro Toshima ◽

Junko Y. Toshima ◽

Mara C. Duncan ◽

M. Jamie T.V. Cope ◽

Yidi Sun ◽

...

Keyword(s):

Actin Polymerization ◽

Affinity Purification ◽

Coiled Coil ◽

Actin Assembly ◽

Related Protein ◽

Loss Of Function ◽

Actin Organization ◽

Coiled Coil Region ◽

Kinase Phosphorylation

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites ∼25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification–tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2ΔCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.

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A direct proof that sole actin dynamics drive membrane deformations

10.1101/307173 ◽

2018 ◽

Author(s):

Camille Simon ◽

Rémy Kusters ◽

Valentina Caorsi ◽

Antoine Allard ◽

Majdouline Abou-Ghali ◽

...

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Cell Function ◽

Turgor Pressure ◽

Direct Proof ◽

Actin Dynamics ◽

Actin Binding ◽

Associated Proteins ◽

Low Tension ◽

Membrane Deformations

AbstractCell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that turn into, for example, filopodia or endocytic intermediates. Actin dynamics and actin-binding proteins are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient, in absence of any membrane-associated proteins, to initiate both inward and outward membrane deformation. With actin polymerization triggered at the membrane of liposomes, we produce inward filopodia-like structures at low tension, while outward endocytosis-like structures are robustly generated regardless of tension. Our results are reminiscent of endocytosis in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where they are always required to overcome the opposing turgor pressure. By combining experimental observations with physical modeling, we propose a mechanism for actin-driven endocytosis under high tension or high pressure conditions.

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FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention

Nucleic Acids Research ◽

10.1093/nar/gkaa410 ◽

2020 ◽

Vol 48 (12) ◽

pp. 6889-6905 ◽

Cited By ~ 7

Author(s):

Jack Humphrey ◽

Nicol Birsa ◽

Carmelo Milioto ◽

Martha McLaughlin ◽

Agnieszka M Ule ◽

...

Keyword(s):

Rna Processing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intron Retention ◽

Mrna Splicing ◽

Loss Of Function ◽

Induced Changes ◽

The Impact ◽

High Depth ◽

Lateral Sclerosis

Abstract Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

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Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration

Blood ◽

10.1182/blood-2012-01-403857 ◽

2013 ◽

Vol 121 (1) ◽

pp. 72-84 ◽

Cited By ~ 11

Author(s):

Austen J. J. Worth ◽

Joao Metelo ◽

Gerben Bouma ◽

Dale Moulding ◽

Marco Fritzsche ◽

...

Keyword(s):

Actin Polymerization ◽

Actin Dynamics ◽

Missense Mutations ◽

Loss Of Function ◽

Interacting Protein ◽

Mutant Proteins ◽

Dendritic Cell Migration ◽

Biologic Function

Abstract Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.

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Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

International Journal of Molecular Sciences ◽

10.3390/ijms21072457 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2457 ◽

Cited By ~ 1

Author(s):

Vikash Singh ◽

Anthony C. Davidson ◽

Peter J. Hume ◽

Vassilis Koronakis

Keyword(s):

Lipid Bilayers ◽

Membrane Trafficking ◽

Actin Polymerization ◽

Small Gtpase ◽

Actin Dynamics ◽

Actin Assembly ◽

Nucleotide Exchange ◽

Cortical Actin ◽

Nucleotide Exchange Factor ◽

Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

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