N-terminal acetylation and arginylation of actin determines the architecture and assembly rate of linear and branched actin networks
2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2022.102518
ISSN1083-351X
AutoresSamantha M. Chin, Tomoyuki Hatano, Lavanya Sivashanmugam, Andrejus Suchenko, Anna Kashina, Mohan K. Balasubramanian, Silvia Jansen,
Tópico(s)Cellular transport and secretion
ResumoThe great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis, and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and cofilin, recent work also implicates posttranslational acetylation or arginylation of the actin N terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing and modification of the N terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in cofilin-mediated severing. Taken together, these results suggest that cells can employ these differently modified actins to regulate actin dynamics. In addition, unprocessed actin with an N-terminal methionine residue shows very different effects on formin-mediated elongation, Arp2/3-mediated nucleation, and severing by cofilin. Altogether, this study shows that the nature of the N terminus of actin can promote distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge. The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis, and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and cofilin, recent work also implicates posttranslational acetylation or arginylation of the actin N terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing and modification of the N terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in cofilin-mediated severing. Taken together, these results suggest that cells can employ these differently modified actins to regulate actin dynamics. In addition, unprocessed actin with an N-terminal methionine residue shows very different effects on formin-mediated elongation, Arp2/3-mediated nucleation, and severing by cofilin. Altogether, this study shows that the nature of the N terminus of actin can promote distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge. Actin cytoskeleton dynamics are the driving force for a myriad of essential cellular functions, including cell migration, cytokinesis, intracellular transport, and contractility (1Blanchoin L. Boujemaa-Paterski R. Sykes C. Plastino J. Actin dynamics, architecture, and mechanics in cell motility.Physiol. Rev. 2014; 94: 235-263Crossref PubMed Scopus (845) Google Scholar, 2Krause M. Gautreau A. Steering cell migration: lamellipodium dynamics and the regulation of directional persistence.Nat. Rev. Mol. Cell Biol. 2014; 15: 577-590Crossref PubMed Scopus (378) Google Scholar, 3Kaksonen M. Roux A. Mechanisms of clathrin-mediated endocytosis.Nat. Rev. Mol. Cell Biol. 2018; 19: 313-326Crossref PubMed Scopus (781) Google Scholar). The functional diversity of actin stems in part from its intrinsic ability to rapidly and reversibly transition between monomeric and filamentous forms and is further expanded through tight spatiotemporal control by hundreds of actin-binding proteins with specific actin-remodeling activities (4Pollard T.D. Actin and actin-binding proteins.Cold Spring Harb Perspect. Biol. 2016; 8: a018226Crossref PubMed Scopus (422) Google Scholar). In addition, it is becoming increasingly clear that direct modification of actin molecules, for example by phosphorylation, acetylation, arginylation, methylation, or oxidation, can have profound effects on actin network dynamics (5A M. Fung T.S. Francomacaro L.M. Huynh T. Kotila T. Svindrych Z. et al.Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 439-447Crossref PubMed Scopus (21) Google Scholar, 6Hung R.-J. Pak C.W. Terman J.R. Direct redox regulation of F-actin assembly and disassembly by mical.Science. 2011; 334: 1710-1713Crossref PubMed Scopus (234) Google Scholar, 7Karakozova M. Kozak M. Wong C.C.L. Bailey A.O. Yates J.R. Mogilner A. et al.Arginylation of ß-actin regulates actin cytoskeleton and cell motility.Science. 2006; 313: 192-196Crossref PubMed Scopus (214) Google Scholar, 8Terman J.R. Kashina A. Post-translational modification and regulation of actin.Curr. Opin. Cell Biol. 2013; 25: 30-38Crossref PubMed Scopus (158) Google Scholar, 9Varland S. Vandekerckhove J. Drazic A. Actin post-translational modifications: the cinderella of cytoskeletal control.Trends Biochem. Sci. 2019; 44: 502-516Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 10Wilkinson A.W. Diep J. Dai S. Liu S. Ooi Y.S. Song D. et al.SETD3 is an actin histidine methyltransferase that prevents primary dystocia.Nature. 2019; 565: 372-376Crossref PubMed Scopus (79) Google Scholar). Modification of actin can occur on many of its exposed residues, including its very N terminus (9Varland S. Vandekerckhove J. Drazic A. Actin post-translational modifications: the cinderella of cytoskeletal control.Trends Biochem. Sci. 2019; 44: 502-516Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The latter is of special interest, as it is the most divergent region in the well-conserved actin molecule, and its modification could be expected to generate unique ways to regulate actin cytoskeleton dynamics. In line with this, posttranslational modification of the N terminus of the abundant cytoplasmic β-actin by mutually exclusive acetylation or arginylation is emerging as a first-line mechanism to regulate cell migration (11Drazic A. Aksnes H. Marie M. Boczkowska M. Varland S. Timmerman E. et al.NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4399-4404Crossref PubMed Scopus (117) Google Scholar, 12Pavlyk I. Leu N.A. Vedula P. Kurosaka S. Kashina A. Rapid and dynamic arginylation of the leading edge β-actin is required for cell migration.Traffic. 2018; 19: 263-272Crossref PubMed Scopus (24) Google Scholar, 13Vedula P. Kurosaka S. MacTaggart B. Ni Q. Papoian G. Jiang Y. et al.Different translation dynamics of β- and γ-actin regulates cell migration.eLife. 2021; 10e68712Crossref PubMed Scopus (16) Google Scholar). Like its cytoplasmic partner γ-actin, the N-terminal initiator Met of β-actin is removed cotranslationally, thereby exposing the second residue (Asp2 in β-actin, Glut2 in γ-actin) for further modification by acetylation (14Arnesen T. Marmorstein R. Dominguez R. Actin's N-terminal acetyltransferase uncovered.Cytoskeleton. 2018; 75: 318-322Crossref Scopus (11) Google Scholar). Although this extensive processing of the N terminus of actin was characterized ∼30 years ago, it was not until recently that it was shown that acetylation of the exposed acidic residue is mediated by a dedicated N-acetyltransferase, NatH/NAA80, which specifically diverged to only target the acidic N terminus of all six human actins (15Goris M. Magin R.S. Foyn H. Myklebust L.M. Varland S. Ree R. et al.Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4405-4410Crossref PubMed Scopus (50) Google Scholar, 16Rebowski G. Boczkowska M. Drazic A. Ree R. Goris M. Arnesen T. et al.Mechanism of actin N-terminal acetylation.Sci. Adv. 2020; 6eaay8793Crossref Scopus (33) Google Scholar). Surprisingly, NAA80 KO cells are hypermotile and show significant increases in the number of filopodia and total F-actin content, whereas in vitro assays show that nonacetylated β/γ-actin displays slower filament elongation than acetylated β/γ-actin, even in the presence of formins (11Drazic A. Aksnes H. Marie M. Boczkowska M. Varland S. Timmerman E. et al.NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4399-4404Crossref PubMed Scopus (117) Google Scholar). This leaves open the question of how acetylation of actin mechanistically contributes to curbing cell migration. The N terminus of the cytoplasmic actins can be further trimmed down to the second acidic residue, which then can be arginylated by the nonspecific arginyltransferase Ate1 (7Karakozova M. Kozak M. Wong C.C.L. Bailey A.O. Yates J.R. Mogilner A. et al.Arginylation of ß-actin regulates actin cytoskeleton and cell motility.Science. 2006; 313: 192-196Crossref PubMed Scopus (214) Google Scholar, 17Wong C.C.L. Xu T. Rai R. Bailey A.O. Yates J.R. Wolf Y.I. et al.Global analysis of posttranslational protein arginylation.PLoS Biol. 2007; 5: e258Crossref PubMed Scopus (120) Google Scholar). Interestingly, arginylation in combination with slower translation leads to immediate proteasomal degradation of γ-actin (18Zhang F. Saha S. Shabalina S.A. Kashina A. Differential arginylation of actin isoforms is regulated by coding sequence–dependent degradation.Science. 2010; 329: 1534-1537Crossref PubMed Scopus (154) Google Scholar). As a result, only arginylated β-actin (hereafter referred to as R-actin) is detected in cells, where it has been shown to specifically relocate to the leading edge upon induction of cell migration (12Pavlyk I. Leu N.A. Vedula P. Kurosaka S. Kashina A. Rapid and dynamic arginylation of the leading edge β-actin is required for cell migration.Traffic. 2018; 19: 263-272Crossref PubMed Scopus (24) Google Scholar). Although less than 1% of the total β-actin population is estimated to be arginylated (19Chen L. Kashina A. Quantification of intracellular N-terminal β-actin arginylation.Sci. Rep. 2019; 9: 16669Crossref PubMed Scopus (14) Google Scholar), making it highly challenging to detect in whole cell lysates (20Drazic A. Timmerman E. Kajan U. Marie M. Varland S. Impens F. et al.The final maturation state of β-actin Involves N-terminal acetylation by NAA80, not N-terminal arginylation by ATE1.J. Mol. Biol. 2022; 434167397Crossref PubMed Scopus (12) Google Scholar); it is possible that local concentrations at the leading edge during active cell migration are much higher and thus exert significant effects on local actin cytoskeleton dynamics. However, both actin as well as various actin-binding proteins can be modified by Ate1, making it difficult to identify what role and which actin-remodeling activities are directly influenced by arginylation of β-actin (R-actin) (17Wong C.C.L. Xu T. Rai R. Bailey A.O. Yates J.R. Wolf Y.I. et al.Global analysis of posttranslational protein arginylation.PLoS Biol. 2007; 5: e258Crossref PubMed Scopus (120) Google Scholar). Also, NAA80 KO cells show a 7-fold increase in R-actin (17Wong C.C.L. Xu T. Rai R. Bailey A.O. Yates J.R. Wolf Y.I. et al.Global analysis of posttranslational protein arginylation.PLoS Biol. 2007; 5: e258Crossref PubMed Scopus (120) Google Scholar, 20Drazic A. Timmerman E. Kajan U. Marie M. Varland S. Impens F. et al.The final maturation state of β-actin Involves N-terminal acetylation by NAA80, not N-terminal arginylation by ATE1.J. Mol. Biol. 2022; 434167397Crossref PubMed Scopus (12) Google Scholar), which supports the hypothesis that acetylation and arginylation of β-actin are mutually exclusive and that the enhanced motility of NAA80 KO cells might at least in part be due to the increased presence of the more positively charged R-actin. This strongly suggests that acetylated (Ac-actin) and R-actin have different intrinsic dynamics and/or interact differently with key actin-regulatory proteins that control cell migration; however, the underlying molecular mechanisms remain unclear to date. Here, we have used pick-ya-actin, a recently established method to produce pure populations of specifically modified human β-actin in Pichia pastoris, to perform the first direct comparison between acetylated and arginylated mammalian β-actin (21Hatano T. Alioto S. Roscioli E. Palani S. Clarke S.T. Kamnev A. et al.Rapid production of pure recombinant actin isoforms in Pichia pastoris.J. Cell Sci. 2018; 131jcs213827PubMed Google Scholar, 22Hatano T. Sivashanmugam L. Suchenko A. Hussain H. Balasubramanian M.K. Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.J. Cell Sci. 2020; 133jcs241406PubMed Google Scholar). To get a better understanding of the contribution of N-terminal modification of β-actin to actin filament dynamics, we also included pure populations of unprocessed actin with an N-terminal methionine (M-actin). Bulk pyrene fluorescence and total internal refection fluorescence microscopy (TIRFM) analysis elucidated that these actins have distinct intrinsic polymerization properties and interact differently with key actin-binding proteins, including profilin, mDia1, Arp2/3, and cofilin. Altogether, this study shows for the first time that proper processing and different N-terminal modifications of actin can alter actin network dynamics and may thusly provide an extra layer of cytoskeletal regulation in cells. The N terminus of β-actin is cotranslationally processed to prepare it for further modification by either acetylation on Asp2 or arginylation on Asp3; however, it remains unclear how this extensive processing and subsequent modification affect the intrinsic self-assembly dynamics of β-actin. To investigate this, Ac-β-actin (Ac-actin), R-β-actin (R-actin), and unprocessed β-actin (M-actin) were recombinantly produced using the recently developed pick-ya-actin system (21Hatano T. Alioto S. Roscioli E. Palani S. Clarke S.T. Kamnev A. et al.Rapid production of pure recombinant actin isoforms in Pichia pastoris.J. Cell Sci. 2018; 131jcs213827PubMed Google Scholar, 22Hatano T. Sivashanmugam L. Suchenko A. Hussain H. Balasubramanian M.K. Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.J. Cell Sci. 2020; 133jcs241406PubMed Google Scholar). Mass spec analysis confirmed that the purified actins consisted of a pure population of actin monomers that underwent the specified modification or different processing of their N terminus (21Hatano T. Alioto S. Roscioli E. Palani S. Clarke S.T. Kamnev A. et al.Rapid production of pure recombinant actin isoforms in Pichia pastoris.J. Cell Sci. 2018; 131jcs213827PubMed Google Scholar, 22Hatano T. Sivashanmugam L. Suchenko A. Hussain H. Balasubramanian M.K. Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.J. Cell Sci. 2020; 133jcs241406PubMed Google Scholar). Next, we compared the polymerization of these different actins using bulk fluorescence assays and TIRFM. Both methods show that Ac-actin polymerizes faster than any of the other tested actins, whereas R-actin was observed to consistently assemble the slowest of all actins (Fig. 1, B–F and Movie S1). TIRFM further enabled us to directly visualize and accurately quantify effects on actin filament nucleation versus actin filament elongation. This analysis showed that spontaneous actin filament nucleation was about 5-fold higher for Ac-actin compared to any of the other actins (Figs. 1, D and E and S1). By contrast, individual actin filaments assembled from each actin were observed to elongate at similar rates (Fig. 1F). Altogether, these independent methods demonstrate that there are clear differences in the intrinsic polymerization and spontaneous nucleation of R-actin and Ac-actin, suggesting that specific modification of the actin N terminus can alter actin filament polymerization. In cells, actin monomers are sequestered by profilin-1 (hereafter referred to as PFN), which curbs spontaneous nucleation and regulates actin filament assembly (23Pollard T.D. Cooper J.A. Quantitative analysis of the effect of acanthamoeba profilin on actin filament nucleation and elongation.Biochemistry. 1984; 23: 6631-6641Crossref PubMed Scopus (239) Google Scholar, 24Rotty J.D. Wu C. Haynes E.M. Suarez C. Winkelman J.D. Johnson H.E. et al.Profilin-1 serves as a gatekeeper for actin assembly by Arp2/3-dependent and -independent pathways.Dev. Cell. 2015; 32: 54-67Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 25Suarez C. Carroll R.T. Burke T.A. Christensen J.R. Bestul A.J. Sees J.A. et al.Profilin regulates F-actin network homeostasis by favoring formin over Arp2/3 complex.Dev. Cell. 2015; 32: 43-53Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Given the differences observed in spontaneous nucleation of Ac-actin and R-actin, we next examined whether modification of the N terminus of actin changes its binding and sequestration by PFN. To this end, actin filaments were polymerized in the presence of increasing concentrations of PFN, pelleted by centrifugation, and analyzed by densitometry. The fraction of filaments in the pellet was used to determine binding of the differently modified actin monomers by PFN (Figs. 2A and S2). This analysis showed that PFN associates similarly with actins with different N-terminal modifications. This is in agreement with a recent structure of monomeric actin bound to PFN and Ac-CoA-NAA80 (16Rebowski G. Boczkowska M. Drazic A. Ree R. Goris M. Arnesen T. et al.Mechanism of actin N-terminal acetylation.Sci. Adv. 2020; 6eaay8793Crossref Scopus (33) Google Scholar), which uniquely enabled to resolve the N terminus of actin. This structure shows that the N terminus of actin is located distally from the PFN binding site and thus should not be affected by N-terminal modification (16Rebowski G. Boczkowska M. Drazic A. Ree R. Goris M. Arnesen T. et al.Mechanism of actin N-terminal acetylation.Sci. Adv. 2020; 6eaay8793Crossref Scopus (33) Google Scholar). Importantly, our data further suggest that in cells, assembly of PFN-bound Ac-actin and R-actin is regulated by additional actin-nucleating proteins. Having established that PFN interacts similarly with Ac-actin and R-actin, we next analyzed whether N-terminal modification of actin affects formin-mediated assembly. Formins are homodimers that stimulate filament nucleation and elongation, preferentially from PFN-bound actin monomers, using their formin homology domains (FH1 and FH2) (26Zimmermann D. Kovar D.R. Feeling the force: formin's role in mechanotransduction.Curr. Opin. Cell Biol. 2019; 56: 130-140Crossref PubMed Scopus (22) Google Scholar). The FH2 domain dimerizes to nucleate actin filaments and remains processively associated with the barbed end of a growing filament, whereas the unstructured FH1 domain binds and positions the PFN-actin complex for addition to the growing filament end. Given that the N terminus is modeled to protrude from the filament (8Terman J.R. Kashina A. Post-translational modification and regulation of actin.Curr. Opin. Cell Biol. 2013; 25: 30-38Crossref PubMed Scopus (158) Google Scholar, 16Rebowski G. Boczkowska M. Drazic A. Ree R. Goris M. Arnesen T. et al.Mechanism of actin N-terminal acetylation.Sci. Adv. 2020; 6eaay8793Crossref Scopus (33) Google Scholar), this suggests that the more positively charged N terminus resulting from arginylation might change the overall charge of the actin filament, thus potentially altering the interaction of formins, such as mDia1. Indeed, bulk pyrene fluorescence assays showed that filament polymerization by mDia1 was severely decreased for R-actin (Fig. 2, B and C). TIRFM further demonstrated that nucleation of Ac-actin by PFN and mDia1 was similar to nucleation of R-actin and about two-fold decreased compared to the nucleation of M-actin (Fig. 2E). Analysis of individual filaments showed slower mDia1-mediated elongation for both Ac-actin and R-actin compared to M-actin filaments (Fig. 2, D and F and Movie S2). Polymerization of R-actin by mDia1 was only slightly slower than mDia1 elongation of Ac-actin (14.0 ± 0.3 subunits/sec/μM versus 19.3 ± 0.5 subunits/sec/μM). However, our bulk pyrene assays suggest that at higher actin concentrations, such as found in cells, this small difference in elongation rate may result in substantially different total actin mass. Altogether, our pyrene fluorescence and TIRFM data indicate that formin-mediated elongation is sensitive to the nature of the actin N terminus. R-actin shows a distinct lamellipodial localization in migrating cells (12Pavlyk I. Leu N.A. Vedula P. Kurosaka S. Kashina A. Rapid and dynamic arginylation of the leading edge β-actin is required for cell migration.Traffic. 2018; 19: 263-272Crossref PubMed Scopus (24) Google Scholar), suggesting that it is not only used for formin-mediated elongation but also participates in the formation of the extensive branched networks underneath the protruding cell membrane. In line with this, the branched actin network at the leading edge of Ate1 KO cells is severely disorganized, although it remains unclear whether this stems from arginylation of β-actin or the Arp2/3 complex, as both were identified as Ate1 targets (17Wong C.C.L. Xu T. Rai R. Bailey A.O. Yates J.R. Wolf Y.I. et al.Global analysis of posttranslational protein arginylation.PLoS Biol. 2007; 5: e258Crossref PubMed Scopus (120) Google Scholar). Our approach with isoform-pure and singly modified actin uniquely enables us to investigate the direct effects of arginylation of β-actin on Arp2/3-mediated actin filament nucleation and branching and get a better insight into the role of arginylation on branched actin network dynamics. Also, it is unknown whether R-actin accumulation at the leading edge can stimulate cell polarization, protrusion, or retraction of the lamella. To this end, we tested the effect of arginylation of the N terminus of actin on Arp2/3 networks using the well-studied and well-characterized GST-VCA domain of NWASP over the less used and understood VCA domains of other nucleation promoting factors. Bulk pyrene fluorescence assays showed that Arp2/3-mediated nucleation of R-actin was severely reduced (Fig. 3, A and B). In addition, M-actin slightly increased Arp2/3-mediated nucleation compared to Ac-actin. Similar trends were observed when analyzing these actins in TIRFM (Fig. 3, C and D). Branch rate analysis showed significantly decreased formation of branches from the sides of filaments that were polymerized from R-actin, whereas M-actin slightly increased Arp2/3 branching (Fig. 3, C and D and Movie S3). Thus, like formin-mediated elongation, Arp2/3-mediated nucleation is hampered by arginylation (R-actin versus Ac-actin and M-actin), suggesting that the charge of the N terminus can alter interaction of actin-binding proteins with actin filaments. However, in case of Arp2/3-mediated nucleation, it is unclear whether this is due to decreased binding of Arp2/3 to arginylated mother filaments or decreased binding of arginylated actin monomers to NWASP. Our observations show that both formin-mediated filament assembly and Arp2/3-mediated filament nucleation are sensitive to N-terminal modification of actin. However, both Arp2/3 as well as formins are bulky molecules that can be more easily affected by changes in the overall charge and surface of actin filaments. Thus, we next tested whether N-terminal modification of actin also changes the interaction of a small actin-binding molecule. For this purpose, we chose the F-actin disassembly factor, cofilin. We directly compared cofilin-mediated severing of Ac-actin and R-actin filaments using TIRFM. Our results show a small reduction in the cumulative severing rate of R-actin compared to Ac-actin; however, this slight decrease did not significantly change the time to half-maximal severing (Fig. 4, B and C and Movie S4). Surprisingly, fragmentation of unprocessed M-actin was enhanced in comparison to Ac-actin and R-actin. Taken together, our data show that interaction of cofilin with physiologically relevant acetylated and arginylated actin filaments is similar in vitro. Combined with our previous results on actin assembly by formins and Arp2/3, this suggests that under cellular conditions, modification of the actin N terminus more likely affects actin network assembly than disassembly. However, our observations for M-actin do leave the possibility that a yet unidentified N-terminal modification may make actin filaments more prone to cofilin-mediated severing. As such, it would be of interest to know whether the enhanced severing seen by M-actin is due to altered cooperative binding of cofilin to the M-actin filament being more prone to the conformational changes induces by cofilin, remains an open question. Our results show that branched and formin-mediated actin networks are assembled differently from pure populations of Ac-actin, and R-actin. However, in cells, both Ac-actin and R-actin localize to the leading edge, which raises the question how actin network assembly is regulated in the presence of both modified actins. To investigate this, we measured formin-mediated assembly and Arp2/3-mediated nucleation of Ac-actin mixed with an increasing percentage (0–60%) of R-actin in bulk pyrene assays. In line with our previous observations, assembly using 100% R-actin monomers was severely reduced compared to assembly from 100% Ac-actin monomers (Fig. 5A). Spiking in 10% R-actin into Ac-actin slightly reduced actin filament assembly compared to 100% Ac-actin, and this effect became more pronounced with increasing percentages of R-actin (Fig. 5A), suggesting that changes in the ratio of R-actin to Ac-actin can alter the rate of actin polymerization. A recent study showed that R-actin can locally accumulate to ∼25% of actin at the leading edge (19Chen L. Kashina A. Quantification of intracellular N-terminal β-actin arginylation.Sci. Rep. 2019; 9: 16669Crossref PubMed Scopus (14) Google Scholar). Thus, we analyzed specifically whether a 1:3 ratio of R-actin to Ac-actin (25% R-actin with 75% Ac-actin) can change actin polymerization in bulk pyrene fluorescence assays. Analysis of the half-times showed that the presence of 25% R-actin did not significantly alter polymerization compared to actin polymerization by Ac-actin only (Fig. 5, B and C). By contrast, slightly different polymerization of Ac-actin was observed at a 1:1 ratio of R-actin to Ac-actin (Fig. 5, B and C). Altogether, these data demonstrate that for cellular actin network polymerization to be affected by R-actin, the local concentration of R-actin needs to increase to or above the concentration of Ac-actin to influence mDia1-mediated elongation and Arp2/3-mediated nucleation. It has been known for many years that actin monomers and filaments can undergo a multitude of modifications; however, the functional impact of many of these modifications remains elusive. Here, we used a recently established system to generate specifically modified actins in Pichia and show that N-terminal modification can have a profound effect on actin filament assembly. Specifically, we focus on directly comparing the effect of arginylation and acetylation of the N terminus of β-actin on actin-binding proteins that shape the actin networks at the leading edge. In addition, we assessed the effects of proper processing of the actin N terminus using a nonphysiological M-actin.
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