Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.
References
[1]
Rajan I, Cline HT. Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo. J Neurosci. 1998;18: 7836–7846. pmid:9742152
[2]
Chevaleyre V, Castillo PE. Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP. Proc Natl Acad Sci U S A. 2002;99: 9538–9543. pmid:12093909 doi: 10.1073/pnas.142213199
[3]
Ohashi R, Sakata S, Naito A, Hirashima N, Tanaka M. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells. Dev Neurobiol. 2014;74: 467–480. doi: 10.1002/dneu.22139. pmid:24123915
[4]
Gaudillière B, Konishi Y, de la Iglesia N, Yao G, Bonni A. A CaMKII-NeuroD signaling pathway specifies dendritic morphogenesis. Neuron. 2004;41: 229–241. pmid:14741104 doi: 10.1016/s0896-6273(03)00841-9
[5]
Fink CC, Bayer KU, Myers JW, Ferrell JE Jr, Schulman H, Meyer T. Selective regulation of neurite extension and synapse formation by the β but not the α isoform of CaMKII. Neuron. 2003;39: 283–297. pmid:12873385 doi: 10.1016/s0896-6273(03)00428-8
[6]
Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T, et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell. 1999;97: 221–231. pmid:10219243 doi: 10.1016/s0092-8674(00)80732-1
[7]
Miki H, Yamaguchi H, Suetsugu S, Takenawa T. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature. 2000;408: 732–735. pmid:11130076 doi: 10.1038/35047107
[8]
Li F, Higgs HN. The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition. Curr Biol. 2003;13: 1335–1340. pmid:12906795 doi: 10.1016/s0960-9822(03)00540-2
[9]
Lammers M, Rose R, Scrima A, Wittinghofer A. The regulation of mDia1 by autoinhibition and its release by Rho*GTP. EMBO J. 2005; 24: 4176–4187. pmid:16292343 doi: 10.1038/sj.emboj.7600879
[10]
Qualmann B, Kessels MM. New players in actin polymerization–WH2-domain-containing actin nucleators. Trends Cell Biol. 2009;19: 276–285. doi: 10.1016/j.tcb.2009.03.004. pmid:19406642
[11]
Campellone KG, Welch MD. A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol. 2010;11: 237–251. doi: 10.1038/nrm2867. pmid:20237478
[12]
Ahuja R, Pinyol R, Reichenbach N, Custer L, Klingensmith J, Kessels MM, et al. Cordon-bleu is an actin nucleation factor and controls neuronal morphology. Cell. 2007;131: 337–350. pmid:17956734 doi: 10.1016/j.cell.2007.08.030
[13]
Firat-Karalar EN, Hsiue PP, Welch MD. The actin nucleation factor JMY is a negative regulator of neuritogenesis. Mol Biol Cell. 2011;22: 4563–4574. doi: 10.1091/mbc.E11-06-0585. pmid:21965285
[14]
Husson C, Renault L, Didry D, Pantaloni D, Carlier MF. Cordon-Bleu uses WH2 domains as multifunctional dynamizers of actin filament assembly. Mol Cell. 2011;43: 464–477. doi: 10.1016/j.molcel.2011.07.010. pmid:21816349
[15]
Haag N, Schwintzer L, Ahuja R, Koch N, Grimm J, Heuer H, et al. The actin nucleator Cobl is crucial for Purkinje cell development and works in close conjunction with the F-actin binding protein Abp1. J Neurosci. 2012;32: 17842–17856. doi: 10.1523/JNEUROSCI.0843-12.2012. pmid:23223303
[16]
Schwintzer L, Koch N, Ahuja R, Grimm J, Kessels MM, Qualmann B. The functions of the actin nucleator Cobl in cellular morphogenesis critically depend on syndapin I. EMBO J. 2011;30: 3147–3159. doi: 10.1038/emboj.2011.207. pmid:21725280
[17]
Sutoo D, Akiyama K, Fujii N, Matsushita K. H-NMR studies of calmodulin: the effect of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene？sulfonamide)and Ca2+ on conformational changes of calmodulin. Biochim Biophys Acta. 1986;873: 156–160. pmid:3741880 doi: 10.1016/0167-4838(86)90203-7
[18]
Norman JA, Ansell J, Stone GA, Wennogle LP, Wasley JWF. CGS 9343B, a novel, potent, and selective inhibitor of calmodulin activity. Mol Pharmacol. 1987;31: 535–540. pmid:3033469
[19]
Minowa O, Yagi K. Calcium binding to tryptic fragments of calmodulin. J Biochem. 1984;96: 1175–1182. pmid:6520119
[20]
Shifman JM, Choi MH, Mihalas S, Mayo SL, Kennedy MB. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums. Proc Natl Acad Sci U S A. 2006;103: 13968–13973. pmid:16966599 doi: 10.1073/pnas.0606433103
[21]
Valeyev NV, Bates DG, Heslop-Harrison P, Postlethwaite I, Kotov NV. Elucidating the mechanisms of cooperative calcium-calmodulin interactions: a structural systems biology approach. BMC Syst Biol. 2008;2: 48–65. doi: 10.1186/1752-0509-2-48. pmid:18518982
[22]
Dharmalingam E, Haeckel A, Pinyol R, Schwintzer L, Koch D, Kessels MM, et al. F-BAR proteins of the syndapin family shape the plasma membrane and are crucial for neuromorphogenesis. J Neurosci. 2009;29: 13315–13327. doi: 10.1523/JNEUROSCI.3973-09.2009. pmid:19846719
[23]
Schneider K, Seemann E, Liebmann L, Ahuja R, Koch D, Westermann M, et al. ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function. J Cell Biol. 2014;205: 197–215. doi: 10.1083/jcb.201307088. pmid:24751538
[24]
Lohmann C. Calcium signaling and the development of specific neuronal connections. Prog Brain Res. 2009;175: 443–452. doi: 10.1016/S0079-6123(09)17529-5. pmid:19660672
[25]
Riederer BM, Zagon IS, Goodman SR. Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells. J Cell Biol. 1986;102: 2088–2097. pmid:3519621 doi: 10.1083/jcb.102.6.2088
[26]
Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM, et al. Competitive binding of α-actinin and calmodulin to the NMDA receptor. Nature. 1997;385: 439–442. pmid:9009191 doi: 10.1038/385439a0
[27]
Hall DD, Dai S, Tseng PY, Malik Z, Nguyen M, Matt L, et al. Competition between α-actinin and Ca2+-calmodulin controls surface retention of the L-type Ca2+ channel CaV1.2. Neuron. 2013;78: 483–497. doi: 10.1016/j.neuron.2013.02.032. pmid:23664615
[28]
Hudmon A, Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function. Annu Rev Biochem. 2002;71: 473–510. pmid:12045104 doi: 10.1146/annurev.biochem.71.110601.135410
[29]
Smart FM, Halpain S. Regulation of dendritic spine stability. Hippocampus. 2000;10: 542–554. pmid:11075824 doi: 10.1002/1098-1063(2000)10:5<542::aid-hipo4>3.0.co;2-7
[30]
Lohmann C, Finski A, Bonhoeffer T. Local calcium transients regulate the spontaneous motility of dendritic filopodia. Nat Neurosci. 2005; 8: 305–312. pmid:15711541 doi: 10.1038/nn1406
[31]
Schüler S, Hauptmann J, Perner B, Kessels MM, Englert C, Qualmann B. Ciliated sensory hair cell formation and function require the F-BAR protein syndapin I and the WH2 domain-based actin nucleator Cobl. J Cell Sci. 2013;126: 196–208. doi: 10.1242/jcs.111674. pmid:23203810
[32]
Qualmann B, Koch D, Kessels MM. Let's go bananas: revisiting the endocytic BAR code. EMBO J. 2011;30: 3501–3515. doi: 10.1038/emboj.2011.266. pmid:21878992
[33]
Kessels MM, Qualmann B. Syndapin oligomers interconnect the machineries for endocytic vesicle formation and actin polymerization. J Biol Chem. 2006;281: 13285–13299. pmid:16540475 doi: 10.1074/jbc.m510226200
[34]
Qualmann B, Roos J, DiGregorio PJ, Kelly RB. Syndapin I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein. Mol Biol Cell. 1999;10: 501–513. pmid:9950691 doi: 10.1091/mbc.10.2.501
[35]
Kessels MM, Engqvist-Goldstein ？EY, Drubin DG. Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation. Mol Biol Cell. 2000;11: 393–412. pmid:10637315 doi: 10.1091/mbc.11.1.393
[36]
Kessels MM, Qualmann B. Syndapins integrate N-WASP in receptor-mediated endocytosis. EMBO J. 2002;21: 6083–6094. pmid:12426380 doi: 10.1093/emboj/cdf604
[37]
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, et al. Lifeact: a versatile marker to visualize F-actin. Nat Methods. 2008;5: 605–607. doi: 10.1038/nmeth.1220. pmid:18536722
[38]
Braun A, Pinyol R, Dahlhaus R, Koch D, Fonarev P, Grant B.D, et al. EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling. Mol Biol Cell. 2005;16: 3642–3658. pmid:15930129 doi: 10.1091/mbc.e05-01-0076
[39]
Qualmann B, Kelly RB. Syndapin isoforms participate in receptor-mediated endocytosis and actin organization. J Cell Biol. 2000;148: 1047–1062. pmid:10704453 doi: 10.1083/jcb.148.5.1047
[40]
Koch N, Dharmalingam E, Westermann M, Qualmann B, Thomas U, Kessels MM. Abp1 utilizes the Arp2/3 complex activator Scar/WAVE in bristle development. J Cell Sci. 2012;125: 3578–3589. pmid:22467854 doi: 10.1242/jcs.101451
[41]
Bers DM, Patton CW, Nuccitelli R. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 1994;40: 3–29. pmid:8201981 doi: 10.1016/s0091-679x(08)61108-5
[42]
Kessels MM, Engqvist-Goldstein ？EY, Drubin DG, Qualmann B. Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin. J. Cell Biol. 2001;153: 351–366. pmid:11309416 doi: 10.1083/jcb.153.2.351
[43]
Qualmann B, Boeckers TM, Jeromin M, Gundelfinger ED, Kessels MM. Linkage of the actin cytoskeleton to the postsynaptic density via direct interactions of Abp1 with the ProSAP/Shank family. J Neurosci. 2004;24: 2481–2495. pmid:15014124 doi: 10.1523/jneurosci.5479-03.2004
[44]
Pinyol R, Haeckel A, Ritter A, Qualmann B, Kessels MM. Regulation of N-WASP and the Arp2/3 complex by Abp1 controls neuronal morphology. PLoS One. 2007;2: e400. pmid:17476322 doi: 10.1371/journal.pone.0000400
