Lysine acetylation of cytoskeletal proteins: Emergence of an actin code.
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ABSTRACT:
SUBMITTER: A M
PROVIDER: S-EPMC7555357 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundFilamin (FLN) and non-muscle alpha-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell.ResultsWe have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD's complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and alpha-actinin (GFP-alphaA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-alphaA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and alpha-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant.ConclusionThese observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton.
| S-EPMC2275727 | biostudies-literature
Project description:INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.
| S-EPMC6955303 | biostudies-literature
Project description:Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys326 and Lys328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin-Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin-Tpm motility that persisted in the presence of Tn and submaximal Ca2+ Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys326 and Lys328, also resulted in less inhibited F-actin-Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn-Tpm-based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys328, modulates muscle contraction via disrupting inhibitory Tpm positioning.
| S-EPMC7667958 | biostudies-literature
Project description:Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.
| S-EPMC8578602 | biostudies-literature
Project description:Myosin II and spectrin ? display mechanosensitive accumulations in invasive protrusions during cell-cell fusion of Drosophila myoblasts. The biochemical inhibition and deactivation of these proteins results in significant fusion defects. Yet, a quantitative understanding of how the protrusion geometry and fusion process are linked to these proteins is still lacking. Here we present a quantitative model to interpret the dependence of the protrusion size and the protrusive force on the mechanical properties and microstructures of the actin cytoskeleton and plasma membrane based on a mean-field theory. We build a quantitative linkage between mechanosensitive accumulation of myosin II and fusion pore formation at the tip of the invasive protrusion through local area dilation. The mechanical feedback loop between myosin II and local deformation suggests that myosin II accumulation possibly reduces the energy barrier and the critical radius of fusion pores. We also analyse the effect of spectrin ? on maintaining the proper geometry of the protrusions required for the success of cell-cell fusion.
| S-EPMC6685025 | biostudies-literature
Project description:Lysine acetylation (Kac) is well known to occur in histones for chromatin function and epigenetic regulation. In addition to histones, Kac is also detected in a large number of proteins with diverse biological functions. However, Kac function and regulatory mechanism for most proteins are unclear. In this work, we studied mutation effects of rice genes encoding cytoplasm-localized histone deacetylases (HDAC) on protein acetylome and found that the HDAC protein HDA714 was a major deacetylase of the rice non-histone proteins including many ribosomal proteins (r-proteins) and translation factors that were extensively acetylated. HDA714 loss-of-function mutations increased Kac levels but reduced abundance of r-proteins. In vitro and in vivo experiments showed that HDA714 interacted with r-proteins and reduced their Kac. Substitutions of lysine by arginine (depleting Kac) in several r-proteins enhance, while mutations of lysine to glutamine (mimicking Kac) decrease their stability in transient expression system. Ribo-seq analysis revealed that the hda714 mutations resulted in increased ribosome stalling frequency. Collectively, the results uncover Kac as a functional posttranslational modification of r-proteins which is controlled by histone deacetylases, extending the role of Kac in gene expression to protein translational regulation.
| S-EPMC8096213 | biostudies-literature
Project description:Many Rickettsia species are intracellular bacterial pathogens that use actin-based motility for spread during infection. However, while other bacteria assemble actin tails consisting of branched networks, Rickettsia assemble long parallel actin bundles, suggesting the use of a distinct mechanism for exploiting actin. To identify the underlying mechanisms and host factors involved in Rickettsia parkeri actin-based motility, we performed an RNAi screen targeting 115 actin cytoskeletal genes in Drosophila cells. The screen delineated a set of four core proteins-profilin, fimbrin/T-plastin, capping protein, and cofilin--as crucial for determining actin tail length, organizing filament architecture, and enabling motility. In mammalian cells, these proteins were localized throughout R. parkeri tails, consistent with a role in motility. Profilin and fimbrin/T-plastin were critical for the motility of R. parkeri but not Listeria monocytogenes. Our results highlight key distinctions between the evolutionary strategies and molecular mechanisms employed by bacterial pathogens to assemble and organize actin.
| S-EPMC2935136 | biostudies-literature
Project description:Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ~180 to 190 nanometers. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin-based cytoskeleton.
| S-EPMC3815867 | biostudies-literature
Project description:Identification of a putative network of actin-associated cytoskeletal proteins in glomerular podocytes defined by co-purified mRNAs. An experimental purification in which a presumed RNA binding protein was used for an RNA immunoprecipitation and microarray analysis (RIP-CHIP). Total cell extracts were harvested from the conditionally immortalized mouse MPC-5 podocyte cell line 14 days after transfer from 33 to 37 degrees Celsius. The MPC-5 cell lines were stably expressing the Wilm's Tumor protein (+KTS) isoform carrying a modified TAP tag. A fraction of each extract (Input samples) was saved and the remaining extract was applied to a sepharose IgG column, washed, and eluates were harvested after addition of TEV protease and elution in microbiospin columns. Four input and four eluate samples were then used to harvest RNA using Trizol. Total RNA was harvested from 4 input samples and 4 eluate samples. RNAs that were enriched in the eluate samples relative to input samples were further investigated and characterized. These RNAs were shown to be enriched in the eluates independent of the TAP-tagged protein. Complete data matrices reporting all 46,657 unique probe IDs (i.e., including the 14 control IDs not reported in the Sample data tables) are linked below as supplementary files.
2009-08-01 | E-GEOD-16942 | biostudies-arrayexpress
Project description:Whereas the neurodegeneration associated with various polyglutamine (polyQ) diseases has prompted extensive studies of polyQ-induced cell death, the neuronal loss that typically appears during late stages of the diseases may not account for the preceding movement and mental disorders. The cellular basis for polyQ-induced neuronal dysfunction preceding neuronal cell death remains largely unknown. Here we report defective dendrite morphogenesis within a specific subset of neurons due to polyQ toxicity that can be dissociated from caspase-dependent cell death. Expressing pathogenic spinocerebellar ataxia type 1 (SCA1) or type 3 (SCA3) proteins in Drosophila larval dendritic arborization neurons caused neuronal type-specific dendrite phenotypes primarily affecting terminal branches. We further show that expression of pathogenic polyQ proteins in adult flies after the formation of neuronal dendrites also greatly reduced dendritic complexity. These defects are associated with disruption of dendritic F-actin structures that can be partially mitigated by increasing Rac-PAK signaling. Together, these findings suggest that specific actin cytoskeletal alterations that alter dendrite morphology and function may contribute to the pathogenesis of at least a subset of polyQ disorders, including SCA3 and SCA1.
| S-EPMC3189048 | biostudies-literature