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Nature Communications Aug 2020EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also...
EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.
Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; Carrier Proteins; Microfilament Proteins; Models, Molecular; Phosphatidylinositol Phosphates; Protein Binding; Protein Conformation; Protein Domains; Protein Interaction Domains and Motifs; Protein Transport; Sequence Alignment; Vesicular Transport Proteins; rab GTP-Binding Proteins
PubMed: 32826901
DOI: 10.1038/s41467-020-17792-3 -
The Journal of Comparative Neurology Sep 2012Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include...
Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain.
Topics: Animals; Blotting, Western; Brain; Contractile Proteins; Filamins; Immunohistochemistry; In Situ Hybridization; Microfilament Proteins; Neurons; Rats; Rats, Sprague-Dawley
PubMed: 22434607
DOI: 10.1002/cne.23106 -
The Journal of Neuroscience : the... Oct 2008Polymerization and organization of actin into complex superstructures, including those found in dendritic spines, is indispensable for structure and function of neuronal... (Comparative Study)
Comparative Study
Polymerization and organization of actin into complex superstructures, including those found in dendritic spines, is indispensable for structure and function of neuronal networks. Here we show that the filamentous actin (F-actin)-binding protein 1 (Abp1), which controls Arp2/3 complex-mediated actin nucleation and binds to postsynaptic scaffold proteins of the ProSAP (proline-rich synapse-associated protein 1)/Shank family, has a profound impact on synaptic organization. Overexpression of the two Abp1 F-actin-binding domains increases the length of thin, filopodia-like and mushroom-type spines but dramatically reduces mushroom spine density, attributable to lack of the Abp1 Src homology 3 (SH3) domain. In contrast, overexpression of full-length Abp1 increases mushroom spine and synapse density. The SH3 domain alone has a dominant-negative effect on mushroom spines, whereas the density of filopodia and thin, immature spines remains unchanged. This suggests that both actin-binding and SH3 domain interactions are crucial for the role of Abp1 in spine maturation. Indeed, Abp1 knockdown significantly reduces mushroom spine and synapse density. Abp1 hereby works in close conjunction with ProSAP1/Shank2 and ProSAP2/Shank3, because Abp1 effects were suppressed by ProSAP2 RNA interference and the ProSAP/Shank-induced increase of spine head width is further promoted by Abp1 cooverexpression and reduced on Abp1 knockdown. Also, interfering with the formation of functional Abp1-ProSAP protein complexes prevents ProSAP-mediated spine head extension. Spine head extension furthermore depends on local Arp2/3 complex-mediated actin polymerization, which is controlled by Abp1 via the Arp2/3 complex activator N-WASP (neural Wiskott-Aldrich syndrome protein). Abp1 thus plays an important role in the formation and morphology control of synapses by making a required functional connection between postsynaptic density components and postsynaptic actin dynamics.
Topics: Actins; Carrier Proteins; Cell Line; Cells, Cultured; Dendritic Spines; Humans; Microfilament Proteins; Protein Binding; Synapses; src Homology Domains
PubMed: 18829961
DOI: 10.1523/JNEUROSCI.0336-08.2008 -
Molecular and Cellular Biology Jun 2005The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal...
The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.
Topics: Actins; Amino Acid Sequence; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cytoskeleton; Fibroblasts; Mice; Microfilament Proteins; Molecular Sequence Data; Morphogenesis; NIH 3T3 Cells; Neurons; Pseudopodia; Recombinant Fusion Proteins; Sequence Alignment; Signal Transduction
PubMed: 15923631
DOI: 10.1128/MCB.25.12.5158-5170.2005 -
Structural plasticity of functional actin: pictures of actin binding protein and polymer interfaces.Structure (London, England : 1993) Oct 2003Actin is one of the most conserved and versatile proteins capable of forming homopolymers and interacting with numerous other proteins in the cell. We performed an...
Actin is one of the most conserved and versatile proteins capable of forming homopolymers and interacting with numerous other proteins in the cell. We performed an alanine mutagenesis scan covering the entire beta-actin molecule. Somewhat surprisingly, the majority of the mutants were capable of reaching a stable conformation. We tested the ability of these mutants to bind to various actin binding proteins, thereby mapping different interfaces with actin. Additionally, we tested their ability to copolymerize with alpha-actin in order to localize regions in actin that contact neighboring protomers in the filament. Hereby, we could discriminate between two existing models for filamentous actin and our data strongly support the right-handed double-stranded helix model. We present data corroborating this model in vivo. Mutants defective in copolymerization do not colocalize with the actin cytoskeleton and some impair its normal function, thereby disturbing cell shape.
Topics: Actins; Cell Cycle Proteins; Cytoskeletal Proteins; Microfilament Proteins; Mutation; Protein Structure, Tertiary; Thymosin
PubMed: 14527395
DOI: 10.1016/j.str.2003.09.002 -
The Journal of Biological Chemistry Jul 2011The microtubule (MT) and actin cytoskeletons are fundamental to cell integrity, because they control a host of cellular activities, including cell division, growth,...
The microtubule (MT) and actin cytoskeletons are fundamental to cell integrity, because they control a host of cellular activities, including cell division, growth, polarization, and migration. Proteins involved in mediating the cross-talk between MT and actin cytoskeletons are key to many cellular processes and play important physiological roles. We identified a new member of the GAS2 family of MT-actin cross-linking proteins, named G2L3 (GAS2-like 3). We show that GAS2-like 3 is widely conserved throughout evolution and is ubiquitously expressed in human tissues. GAS2-like 3 interacts with filamentous actin and MTs via its single calponin homology type 3 domain and C terminus, respectively. Interestingly, the role of the putative MT-binding GAS2-related domain is to modulate the binding of GAS2-like 3 to both filamentous actin and MTs. This is in contrast to GAS2-related domains found in related proteins, where it functions as a MT-binding domain. Furthermore, we show that tubulin acetylation drives GAS2-like 3 localization to MTs and may provide functional insights into the role of GAS2-like 3.
Topics: Actin Cytoskeleton; Animals; Evolution, Molecular; Gene Expression Regulation; HEK293 Cells; HeLa Cells; Humans; Mice; Microfilament Proteins; Microtubule-Associated Proteins; Microtubules; NIH 3T3 Cells; Organ Specificity; Protein Binding; Protein Structure, Tertiary
PubMed: 21561867
DOI: 10.1074/jbc.M111.242263 -
Small GTPases 2015The coronin family of actin-binding proteins regulate actin branching by inhibiting Arp2/3. We recently reported 2 interactions that were unique to coronin-1C: binding...
The coronin family of actin-binding proteins regulate actin branching by inhibiting Arp2/3. We recently reported 2 interactions that were unique to coronin-1C: binding of a Rac1 inhibitor, RCC2, to the unique linker region and Rac1 itself to the propeller domain in a manner that differs from that proposed for other coronins. Through these interactions coronin-1C redistributes Rac1 from the back of the cell to the leading edge for either activation or sequestration by the associated Rac1-inhibitor, RCC2. Here we investigate the relationship between the Rac1- and actin-binding properties of coronin-1C and find that, although actin appears to be involved in the retrafficking of Rac1, signaling by Rac1 lies upstream of the stress fiber-formation, for which the coronins were originally characterized.
Topics: Actins; Amino Acid Sequence; Animals; Humans; Mice; Microfilament Proteins; Molecular Sequence Data; Protein Binding; Protein Transport; Sequence Alignment; rac1 GTP-Binding Protein
PubMed: 25862165
DOI: 10.4161/21541248.2014.992259 -
European Journal of Cell Biology Apr 2022Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for...
Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.
Topics: Actin Cytoskeleton; Actins; Amyotrophic Lateral Sclerosis; Formins; Humans; Microfilament Proteins; Profilins
PubMed: 35248815
DOI: 10.1016/j.ejcb.2022.151212 -
Biochemistry. Biokhimiia Nov 2011Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a...
Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.
Topics: Actin Cytoskeleton; Actins; Androstadienes; Animals; CHO Cells; Cell Membrane; Cortactin; Cricetinae; Cytochalasin D; Gene Knockdown Techniques; Glucose Transporter Type 4; Humans; Insulin; Microfilament Proteins; Muscle Fibers, Skeletal; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Stress Fibers; Wortmannin; src-Family Kinases
PubMed: 22117553
DOI: 10.1134/S0006297911110083 -
Genome Biology 2002The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for... (Review)
Review
The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.
Topics: Actin Depolymerizing Factors; Actins; Animals; Cofilin 2; Destrin; Evolution, Molecular; Exons; Gene Expression Regulation; Humans; Introns; Microfilament Proteins; Multigene Family
PubMed: 12049672
DOI: 10.1186/gb-2002-3-5-reviews3007