© 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 146-152 doi:10.1242/jeb.126383 REVIEW Nebulin, a multi-functional giant Miensheng Chu, Carol C. Gregorio* and Christopher T. Pappas ABSTRACT multi-domain proteins that bind actin filaments via their common Efficient muscle contraction in skeletal muscle is predicated on the nebulin repeats. Notably, all members seem to have roles as regulation of actin filament lengths. In one long-standing model that was stabilizers and/or scaffolds for the highly specialized cytoskeletal prominent for decades, the giant protein nebulin was proposed to assemblies with which they are united with (e.g. sarcomeres, function as a ‘molecular ruler’ to specify the lengths of the thin filaments. intercalated discs and focal adhesions) (for review, see Pappas et al., This theory was questioned by many observations, including 2011). Moreover, increasing evidence links members of the nebulin experiments in which the length of nebulin was manipulated in family to human disease. skeletal myocytes; this approach revealed that nebulin functions to Expression of nebulin outside of skeletal muscle is still debated. stabilize filamentous actin, allowing thin filaments to reach mature Several studies using RT-PCR and immunofluorescence staining lengths. In addition, more recent data, mostly from in vivo models and have demonstrated that nebulin is found (albeit at reduced levels) in identification of new interacting partners, have provided evidence the heart and localizes in the sarcomere, similar to what is observed that nebulin is not merely a structural protein. Nebulin plays a role in skeletal muscle (Bang et al., 2006; Joo et al., 2004; Kazmierski in numerous cellular processes including regulation of muscle et al., 2003). Via the cross-breeding of heterozygous nebulin nebulin contraction, Z-disc formation, and myofibril organization and assembly. mice (where the endogenous gene was replaced by Cre recombinase cDNA) with Rosa26 reporter mice, Bang and KEY WORDS: Actin filament, Sarcomere, Skeletal muscle colleagues further showed that nebulin is predominantly expressed in atrial cardiomyocytes with only minimal expression Introduction in the ventricle (Bang et al., 2006). Reduction of nebulin expression Actin is the prominent protein in most eukaryotic cells. Control of in neonatal cardiac myocytes in culture results in elongation of actin filament organization and architecture is crucial for numerous thin filaments from their pointed ends (McElhinny et al., 2005). cellular functions. The arrangement of actin filaments in striated However, nebulin protein has never been reported in the heart by muscle is quite unique. Individual contractile units of striated immunoblot analysis. Furthermore, global nebulin knockout mice muscle cells (sarcomeres) are composed of overlapping thin (actin) do not exhibit cardiac dysfunction (Bang et al., 2006; Witt et al., and thick (myosin) filaments forming a near-crystalline structure. 2006). Taken together, these results reveal that nebulin is present at Efficient contraction is dependent on the precise regulation of both low levels and could potentially be involved in thin filament length thin and thick filament lengths. A few actin-binding proteins have regulation by a mechanism in which a limited number of nebulin been identified as playing important roles in maintaining thin molecules control the length of many thin filaments in the heart (for filament length; however, the mechanisms that specify thin filament how this may work, see Horowits, 2006). To help resolve whether length have been more elusive. For more than two decades, the huge there is a functional role for nebulin in the heart, further studies protein nebulin (600–900 kD), which is highly expressed in skeletal using cardiac-specific nebulin knockout mice need to be conducted. muscle, was thought to function as a molecular ruler specifying thin For a more detailed description of nebulin and its family members in filament length. Because of the ‘nebulous’ nature of this protein – its the heart, see the recent review by Bang and Chen (2015). susceptibility to proteolysis, enormous size and difficulties in purifying it in its native state – progress in testing the ruler Nebulin interacting partners hypothesis and determining the roles of nebulin in striated muscle The N terminus of nebulin is located near the pointed end of the thin has been slow. filament and contains a high-affinity interacting site for the actin The nebulin family comprises four additional members with filament capping protein tropomodulin (Tmod). Tmod has two diverse localization patterns and cellular roles: N-RAP, nebulette, tropomyosin binding sites, which contribute to the capping lasp-1 and lasp-2. Although each family member interacts with actin efficiency at actin filament pointed ends. It also contains two via their distinguishing ‘nebulin repeats’, each is made up of distinct actin-binding domains that interact with two different sites on actin arrangements of protein motifs (e.g. SH3, LIM) resulting in very filament pointed ends (Boczkowska et al., 2015; Kostyukova et al., diverse molecular sizes (34 to 900 kDa). In particular, the other 2007). Because in mature muscle thin filament pointed ends extend nebulin family members contain fewer nebulin repeats. Nebulette, past the N terminus of nebulin (see next section for discussion), it is lasp-1 and lasp-2 do not have super repeats, while N-RAP has fewer possible that the interaction of nebulin and Tmod is transient, with super repeats than nebulin. With the exception of N-RAP, the other both proteins working together only early in myofibril assembly to family members contain an SH3 domain. Finally, N-RAP, lasp-1 regulate actin filament assembly (McElhinny et al., 2001; Castillo and lasp-2 contain a LIM domain not found in nebulin and et al., 2009). A compelling ‘cap locator’ model of thin filament nebulette. As such, all members of the nebulin family are unique length regulation was proposed by Fowler and coworkers (2006). In this model, nebulin recruits Tmod to a location specified by the Department of Cellular and Molecular Medicine and the Sarver Molecular length of nebulin. Tmod is then able to diffuse and transiently cap Cardiovascular Research Program, The University of Arizona, 1656 East Mabel, thin filaments that are either shorter or longer than nebulin’s length. MRB315, Tucson, AZ 85724, USA. Because of the potential transient nature of the Tmod–nebulin *Author for correspondence ([email protected]) interaction, further studies are required to test whether this model is Journal of Experimental Biology 146 ora fEprmna Biology Experimental of Journal 147 very erminal ent barbed rdigitate lament length 3) contain the , super-repeat – 3 (M1 – -actinin, myopalladin α s C-terminal region, modules 163 ’ COOH SH3 Ser Xin Titin -actinin Zyxin M185 XIRP2 S22). The super repeats are characterized Palladin α M184 N-WASP – Z-disc M183 Myopalladin M182 -actinin, titin, myopalladin, palladin, zyxin, M181 α M180 M179 Titin M178d CapZ (1) M178c M178b M178a M177d M177c Thin filament M177b M177a M176 s linker repeats and this interaction plays a role in M175 ’ M174 M173 M172 s C terminus is located within the highly specialized ’ M171 M170 Z-disc region M169 Nebulin M168 desmin M167 Leiomodin M166 M165 R7) form 22 super repeats (S1 Journal of Experimental Biology (2016) 219, 146-152 doi:10.1242/jeb.126383 M164 – The nebulin SH3 domain in the Z-disc interacts with multiple Nebulin M163 R7 R6 boundary of the sarcomere (Z-disc),myofibril and assembly, plays an mechanosensing, importantand signaling, role in transmission, force and generation sarcolemmalet resilience al., 2002; (reviewed by FrankFig. et Clark al., 1). 2006; Luther, Theinteracts 2009; actin Sheikh with et filament the al.,providing 2007; structural barbed linker stability to end repeatsPappas the et capping sarcomere and al., (Labeit 2008). protein SH3 et Thebinds intermediate al., CapZ domain filament nebulin 2006; protein desmin of also regulation of nebulin, thin filament length, spacingwithin of the adjacent thin Z-disc, filaments and myofibril2011; alignment (Conover Conover and et Gregorio, al.,Based 2009; on Bang the etproposed location al., a 2006; of unique model Witt CapZ in et whichactin binding filaments nebulin al., at cross-links sites the 2006). two Z-disc within adjacent peripheryTesting nebulin, (Pappas this et we al., model 2008) would (Fig.or 1). require super-resolution high-resolution fluorescence immunoelectron microscopy. proteins that, based ondifferent their function, groups. cancytoskeletal be The separated organization into three first and includes group is associated with actin and its ubiquitouslyand expressed cysteine-rich homologue protein(Bang palladin, et (CSRP) Ena/VASP al., familyet 2001; al., members, Chitose 1997; et and Nave et al., zyxin al., 2010; 1990; Li Ma et and al., Wang., 2002; 2004; Moncman Louis R5 R4 R3 S22 The basic contractile units of muscle (sarcomeres) include actin (thin) filaments, CapZ (2) R2 s R1 A-band I-band M-band ’ R7 Sarcomere R6 R5 R4 R3 S21 R2 KLHL40 R1 Tropomodulin R7 Thick filament R6 R5 R4 R3 S1 R2 R1 M8 M7 M6 M5 M4 M3 M2 M1 Neb -actinin N-Term α M162, where seven module repeats (R1 Tropomodulin – NH2 I-band Z-disc . in vivo KO mice and KLHL40-deficient patients, nebulin protein The central super repeat region of nebulin has been demonstrated interaction withcooperatively pointed impact thin end filament lengths. proteins to determine how they with the thick filaments in the A-band, and extend toward the M-line with their pointed ends, where tropomodulin and leiomodin bind to regulate thin fi Fig. 1. The structure ofmyosin nebulin (thick) and filaments, the and location giant of molecules its of binding nebulin partners. and(note titin. that The it thin is filaments not insertregion known in extends whether the near tropomodulin Z-disc the and by pointed leiomodin their end can barbed of bind ends, the to thin span the filament, the same and I-band, thin the inte filament).