- Email: [email protected]
UBE2S Learns Self-Control
Structure
Previews UBE2S Learns Self-Control Tatyana Bodrug1 and Nicholas G. Brown2,* 1Department
of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.07.009 2Department
In this issue of Structure, Liess et al. (2019) demonstrate that the cell cycle regulator UBE2S shuts itself off through autoubiquitination at a conserved lysine residue. Since E2s are at the center of the ubiquitination cascade, this presents a possible regulatory mechanism in a multitude of cellular processes. Ubiquitination is a significant and dynamic form of eukaryotic protein regulation that governs a wide range of cellular processes including cell cycle regulation, DNA repair, and immune response. During the cell cycle, substrate ubiquitination needs to be carried out at the right time and place to ensure the correct ordering of cellular events to avoid homeostatic imbalance. Precise regulation is achieved through the control of 2 E1, more than 30 E2, and more than 600 E3 enzymes (Buetow and Huang, 2016). Lorenz and coworkers demonstrate that the cell cycle regulator UBE2S contains a self-inactivation mechanism that may be applicable to nearly a quarter of the E2s (Liess et al., 2019). Substrate ubiquitination cannot occur haphazardly and errant regulation of ubiquitination results in numerous diseases including cancer (Rape, 2018). Within the overall ubiquitination cascade, E2s are the intermediaries, shuttling ubiquitin (Ub) from the E1 and working with the E3 to ubiquitinate target substrates. RING E3s often promote an activated, closed conformation of E2Ub (‘‘’’ denotes covalent) to facilitate the transfer of Ub from the E2- to an E3-bound substrate. Regulation of this system can occur at multiple levels in the cascade; for example, phosphorylation or conjugation of other ubiquitin-like proteins such as SUMO and NEDD8 (Buetow and Huang, 2016). However, understanding the regulation of E3s has largely been the focus and despite their significance, relatively little is known about the structural mechanisms of E2 regulation. The gigantic, multi-subunit 1.2 MDa E3 ligase known as the anaphase-promoting complex/cyclosome (APC/C) has a well-
established role as the gatekeeper of the cell cycle (Rape, 2018). APC/C ubiquitinates its targets with the assistance of two E2s, UBE2C (UBCH10) and UBE2S, to mark cell cycle proteins with polyubiquitin chains for proteasomal destruction. APC/C recruits and activates UBE2C to initiate substrate polyubiquitination and UBE2S to extend polyubiquitin chains using distinct architectures and surfaces. In brief, UBE2C is recruited by the APC2 WHB, and Ub transfer from UBE2C to substrates is facilitated by the canonical E2Ub binding site on the APC11 RING. UBE2S uses an unprecedented mechanism by which the catalytic ubiquitin-conjugating (UBC) domain of UBE2S binds to another surface of APC2 and the C-terminal extension docks to a groove composed of the APC2 and APC4 subunits. UBE2SUb does not need a RING to adopt the active closed conformation, but rather the APC11 RING positions the substrate-linked Ub to receive another Ub from UBE2S to form a Lys11-linked chain (Brown et al., 2016; Wickliffe et al., 2011). Since these enzymes are important for cell cycle timing, many E2s (including UBE2S) and their corresponding E3s are subject to delicate regulation. Both indirect and direct mechanisms regulating UBE2S function are known to exist, including phosphorylation and proteasomal degradation (Bremm et al., 2010; Craney et al., 2016; Williamson et al., 2009). The latter is postulated to occur through autoubiquitination of a lysine-rich C-terminal extension. In this issue of Structure, Lorenz and colleagues demonstrate that regulation of UBE2S and other E2s deserves more attention as autoubiquitination of the UBC domain results in
autoinhibition but not proteasomal degradation, suggesting a dynamic and responsive self-regulatory mechanism (Liess et al., 2019). During ubiquitination reactions, autoubiquitination of the E2 is readily observed, especially in the absence of an E3. This poses a challenge for the full mechanistic characterization of the enzyme as it is often unclear whether this autoubiquitination event is relevant or a side product of reconstituted reactions. To tackle this problem, Liess et al. (2019) use a suite of methodologies, including in vitro ubiquitination assays, molecular dynamics simulations, NMR, quantitative mass spectrometry, immunoprecipitation, and cell-based assays, to investigate the significance of a specific autoubiquitination event on UBE2S (Liess et al., 2019). The C-terminal extension of UBE2S has been ascribed to be its primary regulatory ubiquitination site. However, these are not the only lysines targeted. Interestingly, a sequence alignment of 34 human E2s, including UBE2S, reveals that a lysine near the active site cysteine (Lys+5) is present in 25% of E2s. Solving additional crystal structures of the catalytic core of UBE2S (UBE2SUBC) revealed flexibility within the active site of UBE2SUBC. Specifically, Lys+5 (Lys100) was positioned in either a Lys+5-in or a Lys+5-out state in which the primary amine of lysine is close to or moved away from the catalytic cysteine (Cys95), respectively. Molecular dynamics simulations demonstrated that the Lys-out state is energetically favored. However, enzyme assays with active and inactive versions of UBE2SUBC show that the ubiquitination of this particular lysine residue occurs through intramolecular
Structure 27, August 6, 2019 ª 2019 Elsevier Ltd. 1185
Structure
Previews
transfer supporting the Lys+5cation? Both APC/C E2s, in state and active site flexiUBE2C and UBE2S, have conserved across bility (Figure 1A). Lys+5 several organisms and thus To understand the impact of would be susceptible to selfautoubiquitination on enzyregulation. matic function, the monoubiMore broadly, the conserquitinated form with a Ub vation of this lysine across molecule conjugated at Lys+5 of UBE2S, referred to as the E2 family suggests that UBE2S-Ub, was purified, valicontrolling autoinhibition is dated by mass spectrometry, relevant to many cellular proand used to assay the different cesses. Indeed, this selfroles of UBE2S in the E1-E2inactivation mechanism was E3 cascade. In summary, it already observed for UBE2T, was found that UBE2S-Ub an E2 vital to the Fanconi could not readily accept anemia pathway for DNA another Ub from the E1 or repair (Machida et al., 2006). form Lys11-linked Ub chains. Furthermore, the creation of Furthermore, the authors autoinhibited E2-Ubs would found that Ube2S-Ub adopts change the pool of available a conformation similar to E2s to cooperate with E3s. the active closed conforAs E2s are in the middle of mation by NMR. However, the cascade and multiple E3s using in vitro enzyme assays use multiple E2s, the regulawith APC/C and full-length tory effects in downstream UBE2S, the autoubiquitination signaling could then be propapattern and the enzymatic acgated rapidly and widely. tivity was nearly identical beE2s and E3s are susceptible Figure 1. Schematic of UBE2S Self-Inactivation Mechanisms and Implications tween the wild-type and the to dynamic changes by (A) UBE2S can exist in Lys+5 -in or Lys+5 -out states. The Lys+5-in conforK+5R variant of UBE2S, sugpost-translational modifica+5 cat mation brings Lys (Lys100) in proximity to the active site Cys (C , Cys95) gesting a significant portion tions and protein-protein infor the intramolecular transfer of Ub. This mechanism is postulated to be conserved in potentially 25% of all E2s. of UBE2S autoubiquitination teractions. Self-regulation, (B) UBE2S can transfer its donor Ub from an active, closed UBE2SUb to (1) occurs on the C-terminal autoinhibition by autoubiquitiLys11 on a substrate-linked Ub to form chains on E3-bound substrates for extension. nation, is yet another layer of proteasomal degradation, (2) Lys+5 inactivating UBE2S by blocking E1A critical question recomplexity to an already mediated recharging of UBE2S-Ub with another donor Ub, or (3) lysines on the C-terminal peptide (CTP) extension of UBE2S marking itself for mained: what is the functional complicated process. Neverdestruction. significance of this autoubitheless, Liess et al. (2019) proquitination of UBE2S at Lys+5 vide a strong foundation for in the cell? After generating a polyclonal UBE2S and possibly other E2s. Similarly future work that is needed to fully appreantibody for UBE2S that can monitor to kinases and phosphatases, DUBs ciate the significance of this regulatory both free and ubiquitinated forms of counteract the activity of E2s and E3s, mechanism. UBE2S and performing co-immunopre- including Lys11-linkages formed by cipitations, the authors found that a APC/C-UBE2S (Bonacci et al., 2018). ACKNOWLEDGMENTS significant portion of UBE2S exists in a Since this Lys+5-linked Ub tag is not for monoubiquitinated form. Tandem mass proteasomal-mediated UBE2S destruc- We apologize to all of the individuals whose work tag mass spectrometry then revealed the tion, one or multiple DUBs could assist was not cited due to limitations. T.B. is supported by NIH T32GM008570. N.G.B is supported by existence of UBE2S automodified at in controlling ubiquitination at an earlier NIH R35GM128855 and UCRF. +5 Lys , suggesting a role for autoinhibition step in the cascade than previously in cells. Intriguingly, total amounts of thought. REFERENCES UBE2S and UBE2S-Ub decreased during This study leaves us with several intermitotic exit. UBE2S decreased because esting questions to be answered in the Bonacci, T., Suzuki, A., Grant, G.D., Stanley, N., of proteasomal destruction independent future. First, what cellular factors, for Cook, J.G., Brown, N.G., and Emanuele, M.J. (2018). Cezanne/OTUD7B is a cell cycle-regulated of the automodification of UBE2S at example, DUBs and APC/C accessibility, deubiquitinase that antagonizes the degradation of +5 Lys (Figure 1B). Taken together, these regulate UBE2S autoubiquitination at APC/C substrates. EMBO J. https://doi.org/10. results imply that the self-inactivation Lys+5 and the C-terminus? Interestingly, 15252/embj.201798701. mechanism is cell cycle regulated. the highest levels of UBE2S autoinhibition Bremm, A., Freund, S.M., and Komander, D. The authors propose the presence of a was observed when APC/C function is (2010). Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hypotential deubiquitinase (DUB) that spe- restrained in mitosis. Second, how is the drolyzed by the deubiquitinase Cezanne. Nat. +5 cifically removes these Ub marks from cell cycle regulated by Lys automodifi- Struct. Mol. Biol. 17, 939–947. 1186 Structure 27, August 6, 2019
Structure
Previews Brown, N.G., VanderLinden, R., Watson, E.R., Weissmann, F., Ordureau, A., Wu, K.P., Zhang, W., Yu, S., Mercredi, P.Y., Harrison, J.S., et al. (2016). Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C. Cell 165, 1440–1453. Buetow, L., and Huang, D.T. (2016). Structural insights into the catalysis and regulation of E3 ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 17, 626–642. Craney, A., Kelly, A., Jia, L., Fedrigo, I., Yu, H., and Rape, M. (2016). Control of APC/C-dependent ubiquitin chain elongation by reversible phosphor-
ylation. Proc. 1540–1545.
Natl.
Acad.
Sci.
USA
113,
Liess, A.K.L., Kucerova, A., Schweimer, K., Yu, L., Roumeliotis, T.I., Diebold, M., Dybkov, O., Sotriffer, C., Urlaub, H., Choudhary, J.S., et al. (2019). Autoinhibition mechanism of the ubiquitinconjugating enzyme UBE2S by autoubiquitination. Structure 27, this issue, 1195–1210. Machida, Y.J., Machida, Y., Chen, Y., Gurtan, A.M., Kupfer, G.M., D’Andrea, A.D., and Dutta, A. (2006). UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation. Mol. Cell 23, 589–596.
Rape, M. (2018). Ubiquitylation at the crossroads of development and disease. Nat. Rev. Mol. Cell Biol. 19, 59–70. Wickliffe, K.E., Lorenz, S., Wemmer, D.E., Kuriyan, J., and Rape, M. (2011). The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781. Williamson, A., Wickliffe, K.E., Mellone, B.G., Song, L., Karpen, G.H., and Rape, M. (2009). Identification of a physiological E2 module for the human anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 106, 18213– 18218.
Deconstructing Actin Waves Min Wu1,* 1Department of Biological Sciences, Centre for Bioimaging Sciences, Mechanobiology Institute, National University of Singapore, Singapore *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.07.010
In this issue of Structure, Jasnin et al. (2019) shows how actin waves on the ventral membrane of Dictyostelium cells propagate by de novo nucleation of oblique filaments that are polarized toward the ventral membrane without a preference to the wave direction. Actin dynamics are central for controlling cell shape and motility. While there has been significant progress in understanding the mechanistic details on the nucleation, elongation, branching, breaking, and depolymerization of individual actin filaments, understanding the higher-order actin assembly in situ remains a challenge. Electron microscopy (EM) serves as powerful tool to visualize the organization of actin networks but often comes with technical caveats. Cryo-EM allows the vitrification of the specimen and visualization of cellular structures in its native context but it is limited by the thickness of the sample to 600 nm (Gerisch and Weber, 2007). With the newly developed in situ cryo-electron tomography (cryo-ET) combined with cryo-focused ion beam (cryo-FIB) milling of frozen-hydrated cells, it is now possible to visualize subcellular structures in the native environments of thicker cells. With these advanced EM techniques, the work by Jasnin et al. (2019) provides an unprecedent view of one of the most complex ultrastructure of actin assembly visualized to date: the traveling actin waves on the ventral
(substrate-attached) surface, using Dictyostelium cells as a model. Dictyostelium is a genus of amoeba that has served as an ideal model system for studying actin dynamics. In fact, actin waves were first discovered in Dictyostelium cells (Vicker, 2002). Since then, actin waves have been observed at the cortex of a wide variety of motile and non-motile cellular systems (Yang and Wu, 2018). They are widely speculated to be fundamental for chemotaxis and cell locomotion and could also reflect more generally the fundamental properties of the cell cortex as an excitable system. Underlying the propagating waves of actin is a micronscale coordinated cycles of actin assembly and disassembly events, whose phase differences give the impression of waves. They are distinct from actin flows that are governed by the physical movement of the actin in the direction of the flow. Two potential mechanisms are consistent with actin wave propagation: de novo nucleation of filaments at the wave front or elongation of pre-existing filaments into the direction of wave propagation. To address this question, Jasnin et al. (2019) elegantly mapped the architecture
of the actin network in different phases of actin waves. They used correlative cryo-fluorescence and cryo-FIB milling to identify actin waves within cells. By comparing actin waves with regions outside of the waves and the inner territories, they provide compelling evidences that actin waves propagate through the first mechanism, de novo nucleation of filaments at the wave front, and rule out the possibility of actin filaments arranged in a way that is polarized toward the direction of wave propagation. Actin waves have striking increases in network thicknesses and increased clusters of oblique filaments (as opposed to horizontal meshworks outside) (Figure 1A). To understand the origin of these oblique filaments, it is essential to determine the polarity of individual filaments as well as their lineages. The authors focus on the filaments associated with the Arp2/3 complex, the nucleator of the branches of actin filaments which are abundantly present in the waves (Jasnin et al., 2019). With the recent advance of the Volta phase plate combined with direct electron detection, they were able to identify Arp2/3 complex at the branches of actin filaments and
Structure 27, August 6, 2019 ª 2019 Elsevier Ltd. 1187
Previews UBE2S Learns Self-Control Tatyana Bodrug1 and Nicholas G. Brown2,* 1Department
of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.07.009 2Department
In this issue of Structure, Liess et al. (2019) demonstrate that the cell cycle regulator UBE2S shuts itself off through autoubiquitination at a conserved lysine residue. Since E2s are at the center of the ubiquitination cascade, this presents a possible regulatory mechanism in a multitude of cellular processes. Ubiquitination is a significant and dynamic form of eukaryotic protein regulation that governs a wide range of cellular processes including cell cycle regulation, DNA repair, and immune response. During the cell cycle, substrate ubiquitination needs to be carried out at the right time and place to ensure the correct ordering of cellular events to avoid homeostatic imbalance. Precise regulation is achieved through the control of 2 E1, more than 30 E2, and more than 600 E3 enzymes (Buetow and Huang, 2016). Lorenz and coworkers demonstrate that the cell cycle regulator UBE2S contains a self-inactivation mechanism that may be applicable to nearly a quarter of the E2s (Liess et al., 2019). Substrate ubiquitination cannot occur haphazardly and errant regulation of ubiquitination results in numerous diseases including cancer (Rape, 2018). Within the overall ubiquitination cascade, E2s are the intermediaries, shuttling ubiquitin (Ub) from the E1 and working with the E3 to ubiquitinate target substrates. RING E3s often promote an activated, closed conformation of E2Ub (‘‘’’ denotes covalent) to facilitate the transfer of Ub from the E2- to an E3-bound substrate. Regulation of this system can occur at multiple levels in the cascade; for example, phosphorylation or conjugation of other ubiquitin-like proteins such as SUMO and NEDD8 (Buetow and Huang, 2016). However, understanding the regulation of E3s has largely been the focus and despite their significance, relatively little is known about the structural mechanisms of E2 regulation. The gigantic, multi-subunit 1.2 MDa E3 ligase known as the anaphase-promoting complex/cyclosome (APC/C) has a well-
established role as the gatekeeper of the cell cycle (Rape, 2018). APC/C ubiquitinates its targets with the assistance of two E2s, UBE2C (UBCH10) and UBE2S, to mark cell cycle proteins with polyubiquitin chains for proteasomal destruction. APC/C recruits and activates UBE2C to initiate substrate polyubiquitination and UBE2S to extend polyubiquitin chains using distinct architectures and surfaces. In brief, UBE2C is recruited by the APC2 WHB, and Ub transfer from UBE2C to substrates is facilitated by the canonical E2Ub binding site on the APC11 RING. UBE2S uses an unprecedented mechanism by which the catalytic ubiquitin-conjugating (UBC) domain of UBE2S binds to another surface of APC2 and the C-terminal extension docks to a groove composed of the APC2 and APC4 subunits. UBE2SUb does not need a RING to adopt the active closed conformation, but rather the APC11 RING positions the substrate-linked Ub to receive another Ub from UBE2S to form a Lys11-linked chain (Brown et al., 2016; Wickliffe et al., 2011). Since these enzymes are important for cell cycle timing, many E2s (including UBE2S) and their corresponding E3s are subject to delicate regulation. Both indirect and direct mechanisms regulating UBE2S function are known to exist, including phosphorylation and proteasomal degradation (Bremm et al., 2010; Craney et al., 2016; Williamson et al., 2009). The latter is postulated to occur through autoubiquitination of a lysine-rich C-terminal extension. In this issue of Structure, Lorenz and colleagues demonstrate that regulation of UBE2S and other E2s deserves more attention as autoubiquitination of the UBC domain results in
autoinhibition but not proteasomal degradation, suggesting a dynamic and responsive self-regulatory mechanism (Liess et al., 2019). During ubiquitination reactions, autoubiquitination of the E2 is readily observed, especially in the absence of an E3. This poses a challenge for the full mechanistic characterization of the enzyme as it is often unclear whether this autoubiquitination event is relevant or a side product of reconstituted reactions. To tackle this problem, Liess et al. (2019) use a suite of methodologies, including in vitro ubiquitination assays, molecular dynamics simulations, NMR, quantitative mass spectrometry, immunoprecipitation, and cell-based assays, to investigate the significance of a specific autoubiquitination event on UBE2S (Liess et al., 2019). The C-terminal extension of UBE2S has been ascribed to be its primary regulatory ubiquitination site. However, these are not the only lysines targeted. Interestingly, a sequence alignment of 34 human E2s, including UBE2S, reveals that a lysine near the active site cysteine (Lys+5) is present in 25% of E2s. Solving additional crystal structures of the catalytic core of UBE2S (UBE2SUBC) revealed flexibility within the active site of UBE2SUBC. Specifically, Lys+5 (Lys100) was positioned in either a Lys+5-in or a Lys+5-out state in which the primary amine of lysine is close to or moved away from the catalytic cysteine (Cys95), respectively. Molecular dynamics simulations demonstrated that the Lys-out state is energetically favored. However, enzyme assays with active and inactive versions of UBE2SUBC show that the ubiquitination of this particular lysine residue occurs through intramolecular
Structure 27, August 6, 2019 ª 2019 Elsevier Ltd. 1185
Structure
Previews
transfer supporting the Lys+5cation? Both APC/C E2s, in state and active site flexiUBE2C and UBE2S, have conserved across bility (Figure 1A). Lys+5 several organisms and thus To understand the impact of would be susceptible to selfautoubiquitination on enzyregulation. matic function, the monoubiMore broadly, the conserquitinated form with a Ub vation of this lysine across molecule conjugated at Lys+5 of UBE2S, referred to as the E2 family suggests that UBE2S-Ub, was purified, valicontrolling autoinhibition is dated by mass spectrometry, relevant to many cellular proand used to assay the different cesses. Indeed, this selfroles of UBE2S in the E1-E2inactivation mechanism was E3 cascade. In summary, it already observed for UBE2T, was found that UBE2S-Ub an E2 vital to the Fanconi could not readily accept anemia pathway for DNA another Ub from the E1 or repair (Machida et al., 2006). form Lys11-linked Ub chains. Furthermore, the creation of Furthermore, the authors autoinhibited E2-Ubs would found that Ube2S-Ub adopts change the pool of available a conformation similar to E2s to cooperate with E3s. the active closed conforAs E2s are in the middle of mation by NMR. However, the cascade and multiple E3s using in vitro enzyme assays use multiple E2s, the regulawith APC/C and full-length tory effects in downstream UBE2S, the autoubiquitination signaling could then be propapattern and the enzymatic acgated rapidly and widely. tivity was nearly identical beE2s and E3s are susceptible Figure 1. Schematic of UBE2S Self-Inactivation Mechanisms and Implications tween the wild-type and the to dynamic changes by (A) UBE2S can exist in Lys+5 -in or Lys+5 -out states. The Lys+5-in conforK+5R variant of UBE2S, sugpost-translational modifica+5 cat mation brings Lys (Lys100) in proximity to the active site Cys (C , Cys95) gesting a significant portion tions and protein-protein infor the intramolecular transfer of Ub. This mechanism is postulated to be conserved in potentially 25% of all E2s. of UBE2S autoubiquitination teractions. Self-regulation, (B) UBE2S can transfer its donor Ub from an active, closed UBE2SUb to (1) occurs on the C-terminal autoinhibition by autoubiquitiLys11 on a substrate-linked Ub to form chains on E3-bound substrates for extension. nation, is yet another layer of proteasomal degradation, (2) Lys+5 inactivating UBE2S by blocking E1A critical question recomplexity to an already mediated recharging of UBE2S-Ub with another donor Ub, or (3) lysines on the C-terminal peptide (CTP) extension of UBE2S marking itself for mained: what is the functional complicated process. Neverdestruction. significance of this autoubitheless, Liess et al. (2019) proquitination of UBE2S at Lys+5 vide a strong foundation for in the cell? After generating a polyclonal UBE2S and possibly other E2s. Similarly future work that is needed to fully appreantibody for UBE2S that can monitor to kinases and phosphatases, DUBs ciate the significance of this regulatory both free and ubiquitinated forms of counteract the activity of E2s and E3s, mechanism. UBE2S and performing co-immunopre- including Lys11-linkages formed by cipitations, the authors found that a APC/C-UBE2S (Bonacci et al., 2018). ACKNOWLEDGMENTS significant portion of UBE2S exists in a Since this Lys+5-linked Ub tag is not for monoubiquitinated form. Tandem mass proteasomal-mediated UBE2S destruc- We apologize to all of the individuals whose work tag mass spectrometry then revealed the tion, one or multiple DUBs could assist was not cited due to limitations. T.B. is supported by NIH T32GM008570. N.G.B is supported by existence of UBE2S automodified at in controlling ubiquitination at an earlier NIH R35GM128855 and UCRF. +5 Lys , suggesting a role for autoinhibition step in the cascade than previously in cells. Intriguingly, total amounts of thought. REFERENCES UBE2S and UBE2S-Ub decreased during This study leaves us with several intermitotic exit. UBE2S decreased because esting questions to be answered in the Bonacci, T., Suzuki, A., Grant, G.D., Stanley, N., of proteasomal destruction independent future. First, what cellular factors, for Cook, J.G., Brown, N.G., and Emanuele, M.J. (2018). Cezanne/OTUD7B is a cell cycle-regulated of the automodification of UBE2S at example, DUBs and APC/C accessibility, deubiquitinase that antagonizes the degradation of +5 Lys (Figure 1B). Taken together, these regulate UBE2S autoubiquitination at APC/C substrates. EMBO J. https://doi.org/10. results imply that the self-inactivation Lys+5 and the C-terminus? Interestingly, 15252/embj.201798701. mechanism is cell cycle regulated. the highest levels of UBE2S autoinhibition Bremm, A., Freund, S.M., and Komander, D. The authors propose the presence of a was observed when APC/C function is (2010). Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hypotential deubiquitinase (DUB) that spe- restrained in mitosis. Second, how is the drolyzed by the deubiquitinase Cezanne. Nat. +5 cifically removes these Ub marks from cell cycle regulated by Lys automodifi- Struct. Mol. Biol. 17, 939–947. 1186 Structure 27, August 6, 2019
Structure
Previews Brown, N.G., VanderLinden, R., Watson, E.R., Weissmann, F., Ordureau, A., Wu, K.P., Zhang, W., Yu, S., Mercredi, P.Y., Harrison, J.S., et al. (2016). Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C. Cell 165, 1440–1453. Buetow, L., and Huang, D.T. (2016). Structural insights into the catalysis and regulation of E3 ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 17, 626–642. Craney, A., Kelly, A., Jia, L., Fedrigo, I., Yu, H., and Rape, M. (2016). Control of APC/C-dependent ubiquitin chain elongation by reversible phosphor-
ylation. Proc. 1540–1545.
Natl.
Acad.
Sci.
USA
113,
Liess, A.K.L., Kucerova, A., Schweimer, K., Yu, L., Roumeliotis, T.I., Diebold, M., Dybkov, O., Sotriffer, C., Urlaub, H., Choudhary, J.S., et al. (2019). Autoinhibition mechanism of the ubiquitinconjugating enzyme UBE2S by autoubiquitination. Structure 27, this issue, 1195–1210. Machida, Y.J., Machida, Y., Chen, Y., Gurtan, A.M., Kupfer, G.M., D’Andrea, A.D., and Dutta, A. (2006). UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation. Mol. Cell 23, 589–596.
Rape, M. (2018). Ubiquitylation at the crossroads of development and disease. Nat. Rev. Mol. Cell Biol. 19, 59–70. Wickliffe, K.E., Lorenz, S., Wemmer, D.E., Kuriyan, J., and Rape, M. (2011). The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781. Williamson, A., Wickliffe, K.E., Mellone, B.G., Song, L., Karpen, G.H., and Rape, M. (2009). Identification of a physiological E2 module for the human anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 106, 18213– 18218.
Deconstructing Actin Waves Min Wu1,* 1Department of Biological Sciences, Centre for Bioimaging Sciences, Mechanobiology Institute, National University of Singapore, Singapore *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.07.010
In this issue of Structure, Jasnin et al. (2019) shows how actin waves on the ventral membrane of Dictyostelium cells propagate by de novo nucleation of oblique filaments that are polarized toward the ventral membrane without a preference to the wave direction. Actin dynamics are central for controlling cell shape and motility. While there has been significant progress in understanding the mechanistic details on the nucleation, elongation, branching, breaking, and depolymerization of individual actin filaments, understanding the higher-order actin assembly in situ remains a challenge. Electron microscopy (EM) serves as powerful tool to visualize the organization of actin networks but often comes with technical caveats. Cryo-EM allows the vitrification of the specimen and visualization of cellular structures in its native context but it is limited by the thickness of the sample to 600 nm (Gerisch and Weber, 2007). With the newly developed in situ cryo-electron tomography (cryo-ET) combined with cryo-focused ion beam (cryo-FIB) milling of frozen-hydrated cells, it is now possible to visualize subcellular structures in the native environments of thicker cells. With these advanced EM techniques, the work by Jasnin et al. (2019) provides an unprecedent view of one of the most complex ultrastructure of actin assembly visualized to date: the traveling actin waves on the ventral
(substrate-attached) surface, using Dictyostelium cells as a model. Dictyostelium is a genus of amoeba that has served as an ideal model system for studying actin dynamics. In fact, actin waves were first discovered in Dictyostelium cells (Vicker, 2002). Since then, actin waves have been observed at the cortex of a wide variety of motile and non-motile cellular systems (Yang and Wu, 2018). They are widely speculated to be fundamental for chemotaxis and cell locomotion and could also reflect more generally the fundamental properties of the cell cortex as an excitable system. Underlying the propagating waves of actin is a micronscale coordinated cycles of actin assembly and disassembly events, whose phase differences give the impression of waves. They are distinct from actin flows that are governed by the physical movement of the actin in the direction of the flow. Two potential mechanisms are consistent with actin wave propagation: de novo nucleation of filaments at the wave front or elongation of pre-existing filaments into the direction of wave propagation. To address this question, Jasnin et al. (2019) elegantly mapped the architecture
of the actin network in different phases of actin waves. They used correlative cryo-fluorescence and cryo-FIB milling to identify actin waves within cells. By comparing actin waves with regions outside of the waves and the inner territories, they provide compelling evidences that actin waves propagate through the first mechanism, de novo nucleation of filaments at the wave front, and rule out the possibility of actin filaments arranged in a way that is polarized toward the direction of wave propagation. Actin waves have striking increases in network thicknesses and increased clusters of oblique filaments (as opposed to horizontal meshworks outside) (Figure 1A). To understand the origin of these oblique filaments, it is essential to determine the polarity of individual filaments as well as their lineages. The authors focus on the filaments associated with the Arp2/3 complex, the nucleator of the branches of actin filaments which are abundantly present in the waves (Jasnin et al., 2019). With the recent advance of the Volta phase plate combined with direct electron detection, they were able to identify Arp2/3 complex at the branches of actin filaments and
Structure 27, August 6, 2019 ª 2019 Elsevier Ltd. 1187