- Email: [email protected]
Nebulin: Does It Measure up as a Ruler?
Current Biology Vol 16 No 1 R18
‘minimal’ model in olfactory research. References 1. Fishilevich, E., Domingos, A.I., Asahina, K., Naef, F., Vosshall, L.B., and Louis, M. (2005). Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila. Curr. Biol. 15, 2086-2096. 2. Buck, L., and Axel, R. (1991). A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175–187. 3. Clyne, P.J., Warr, C.G., Freeman, M.R., Lessing, D., Kim, J., and Carlson, J.R. (1999). A novel family of divergent seventransmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22, 327–338. 4. Vosshall, L.B., Amrein, H., Morozov, P.S., Rzhetsky, A., and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96, 725–736. 5. Ressler, K.J., Sullivan, S.L., and Buck, L.B. (1994). Information coding in the olfactory system: Evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245–1255. 6. Vassar, R., Chao, S.K., Sitcheran, R., Nunez, J.M., Vosshall, L.B., and Axel, R. (1994). Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981–991. 7. Gao, Q., Yuan, B., and Chess, A. (2000).
Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat. Neurosci. 3, 780–785. 8. Vosshall, L.B., Wong, A.M., and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102, 147–159. 9. Joerges, J., Küttner, A., Galizia, C.G., and Menzel, R. (1997). Representations of odours and odour mixtures visualized in the honeybee brain. Nature 387, 285–287. 10. Malnic, B., Hirono, J., Sato, T., and Buck, L.B. (1999). Combinatorial receptor codes for odors. Cell 96, 713–726. 11. Wang, J.W., Wong, A.M., Flores, J., Vosshall, L.B., and Axel, R. (2003). Twophoton calcium imaging reveals an odorevoked map of activity in the fly brain. Cell 112, 271–282. 12. Hildebrand, J.G., and Shepherd, G. (1997). Mechanisms of olfactory discrimination: converging evidence for common principles across phyla. Annu. Rev. Neurosci. 20, 595–631. 13. Robertson, H.M., Warr, C.G., and Carlson, J.R. (2003). Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 100, 14537–14542. 14. Couto, A., Alenius, M., and Dickson, B.J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15, 1535–1547. 15. Kreher, S.A., Kwon, A.Y., and Carlson,
Nebulin: Does It Measure up as a Ruler? A recent study has shown that the giant protein nebulin maintains the lengths of actin filaments in striated muscle cells. Although on the surface, nebulin looks like a molecular ruler, it may be playing a more complex role in regulating dynamics at the pointed end of actin filaments in striated muscle. Velia M. Fowler, Caroline R. McKeown and Robert S. Fischer In a striated muscle cell, the lengths of the sarcomeric actin filaments — the thin filaments — are precisely regulated, both in terms of their particular lengths, and in the variation of their length distributions [1,2]. Actin monomers assemble in vitro into filaments with an exponential length distribution, and they can organize into a wide variety of lengths in non-muscle cells, indicating that length controls are not intrinsic to actin filament polymers. Historically, a ‘molecular ruler’ mechanism has been the most attractive model for how thin filament lengths are determined in striated muscle. By definition, a
molecular ruler must meet a number of criteria: the ruler must be the precise length of the target filament, with the length of the ruler dictating the length of the filaments; the end of the ruler should bind to a terminator protein to prevent actin subunit addition or loss once the filament has polymerized to the length of the ruler protein; and the ruler must associate in a stoichiometric ratio with its target filament. In this mechanism, filament lengths are fixed precisely to the ruler length once they have polymerized. Thus, only filaments associated with rulers will be the length of the ruler, and filaments without a ruler will assume random and variable lengths (Figure 1A). The giant molecule nebulin has been postulated to be a molecular
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J.R. (2005). The molecular basis of odor coding in the Drosophila larva. Neuron 46, 445–456. Larsson, M.C., Domingos, A.I., Jones, W.D., Chiappe, M.E., Amrein, H., and Vosshall, L.B. (2004). Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43, 703–714. Hallem, E.A., Ho, M.G., and Carlson, J.R. (2004). The molecular basis of odor coding in the Drosophila antenna. Cell 117, 965–979. Goldman, A.L., van der Goes van Naters, W., Lessing, D., Warr, C.G., and Carlson, J.R. (2005). Coexpression of two functional odor receptors in one neuron. Neuron 45, 661–666. Ramaekers, A., Magnenat, E., Marin, E.C., Gendre, N., Jefferis, G.S.X.E., Luo, L., and Stocker, R.F. (2005). Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit. Curr. Biol. 15, 982–992. Wilson, R.I., Turner, G.C., and Laurent, G. (2004). Transformation of olfactory representations in the Drosophila antennal lobe. Science 303, 366–370.
Department of Biology, University of Fribourg, 10 rue du Musée, CH-1700 Fribourg, Switzerland. DOI: 10.1016/j.cub.2005.12.008
ruler that determines thin filament length in striated muscle [3–7]. Nebulin extends along the thin filament, with its amino terminus oriented near the pointed (free) end and its carboxyl terminus near the barbed end in the Z disc. A number of properties of nebulin appear to fulfill many of the a priori requirements for a molecular ruler. First, the molecular sizes of nebulin isoforms correlate with the lengths of thin filaments in the striated muscles in which the isoform is found [6,8]. Second, nebulin molecules are composed of a modular series of repeats corresponding to the repeats of the actin subunits of the thin filament, thus ‘measuring’ polymer length [6,9]. Third, a region in the unique amino-terminal domain of nebulin (M1M2M3) is located near the thin filament pointed end [9], and interacts with the actin pointed end capping protein, tropomodulin [10,11], thus potentially providing a mechanism to arrest filament elongation at precisely the length of the nebulin template [7] (Figure 1A). In a recent study, Gregorio and colleagues [12] have shown for the first time that nebulin regulates thin filament length. Using RNA
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Figure 1. Models for B Cap locator A Ruler regulation of thin filament Z Z lengths in striated muscle. (A) The ruler mechanism requires that actin subunits polymerize into filaments alongside the nebulin ruler. Actin assembly is terminated at the length of the ruler by tropomodulin (Tmod), which binds tightly to the actin pointed end and to the M1M2M3 domain of nebulin. Regulation of filament length is stoichiometric, requiring each filament to be associated with a nebulin ruler and tightly capped at its pointed end by tropomodNebulin Actin ulin. When filaments are M1M2M3 domain Tmod not associated with rulers, Current Biology for example when nebulin levels are reduced or absent, then filaments are not length regulated and randomly assume both shorter and longer lengths (middle and bottom). Filament lengths are not influenced by increases in the concentration of tropomodulin nor by rates of actin monomer association and dissociation at filament ends. (B) The cap locator mechanism depends on localized regulation of tropomodulin capping and actin dynamics. Dynamic association of tropomodulin with the M1M2M3 domain of nebulin generates an increased local concentration of tropomodulin at a distance from the Z disc (Z) determined by the length of the nebulin. Increased tropomodulin capping of pointed ends in this location competes for actin subunit addition and controls lengths. Lengths do not depend on precise stoichiometric ratios of filaments to rulers, and are sensitive to both tropomodulin concentration and actin dynamics. When nebulin levels are reduced or absent, the local tropomodulin concentration falls, allowing actin subunit addition and graded and uniform elongation of filaments. The precision of length regulation (variability) depends on the relative local concentration of tropomodulin, which depends on nebulin levels and on the affinity of tropomodulin for the M1M2M3 domain of nebulin [11]. Decreasing amount of nebulin
interference technology, nebulin levels were knocked down in cultured rat cardiac myocytes and sarcomere organization and thin filament lengths were evaluated by fluorescence microscopy in both fixed and living cells. Strikingly, after reduction of nebulin levels, actin and tropomyosin were found to extend across the gap in the middle of the sarcomere, indicating that thin filaments had elongated from their pointed ends. No changes were observed in Z disc distances or in the striated organization of titin or thick filaments, demonstrating that sarcomeres were not hypercontracted. Further, tropomodulin was no longer associated with sarcomeres, showing that it had dissociated from pointed ends of the aberrantly elongated thin filaments, as expected if the capping of thin filaments by tropomodulin depends on nebulin. The observed dependence of tropomodulin localization and thin filament length on nebulin levels are both consistent with the idea that nebulin could be a thin filament molecular ruler. Certain results in this study [12], however, lead to the conclusion that nebulin does not regulate length by a simple physical ruler mechanism. At intermediate times, sarcomeres with partially reduced staining for nebulin displayed narrower gaps in actin staining in the middle of the sarcomere. Line scans of the fluorescence intensity across the sarcomeres showed that partial reductions in nebulin led to uniform elongation of thin filaments in individual sarcomeres by up to 30%. This is completely inconsistent with a molecular ruler function for nebulin. If nebulin were a true ruler, the filaments without nebulin would have been expected to assume random lengths, while filaments with nebulin should have remained at their original length (Figure 1A, middle). Instead, fewer nebulins led to graded and uniform increases in filament length (Figure 1B, middle). Comparison of nebulin levels and thin filament lengths in intact muscles also reveals difficulties with a molecular ruler mechanism. While nebulin is present in skeletal
muscle at stoichiometric levels of two per filament [3,7], it is considerably less abundant in cardiac muscle [4,13] where the lengths of thin filaments are still regulated [2,14]. Moreover, myofibrils isolated from some skeletal muscle types have thin filament lengths that vary by about 100 nm [14], despite the predominance of one nebulin isoform in each muscle type [8]. Significantly, a key feature of thin filament length control that is not explained by a molecular ruler mechanism is the role of actin dynamics. Lengths of muscle thin filaments are controlled by regulation of actin assembly at the pointed, but not barbed, filament ends. The pointed ends of actin filaments in muscle are transiently capped by tropomodulin, allowing for continuous actin subunit exchange [15]. An increase in the tropomodulin level reduces actin incorporation at pointed ends and leads to uniform shortening of thin filaments [15–17]. Conversely, a
decrease in the tropomodulin level or inhibition of tropomodulin’s actin-capping activity results in thin filament elongation [16,18,19]. While these experiments demonstrate that regulation of actin assembly dynamics by tropomodulin controls filament lengths, determination of specific lengths requires that rates of assembly must be lengthdependent [2,5,20]. We propose that nebulin may be a ‘cap locator’ that provides a position-dependent regulation of tropomodulin capping to determine thin filament lengths (Figure 1B). In the simplest mechanism, dynamic interaction of tropomodulin with the aminoterminal M1M2M3 region of nebulin near the pointed end could serve to create a localized concentration of tropomodulin, at a distance from the Z disc determined by the length of nebulin, as suggested previously by Marshall [5]. In this mechanism, nebulin molecules
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need not be stoichiometric with each thin filament, nor does nebulin regulate filament lengths in a binary, all-or-nothing fashion. As the numbers of nebulins per sarcomere are decreased, the local concentration of tropomodulin is diminished, leading to lower effective capping activity, increased actin assembly and uniformly longer thin filaments (Figure 1B). This model is also consistent with the dependence of thin filament length on tropomodulin concentrations [15–17]. How can the question of whether nebulin acts as a molecular ruler or a cap locator be resolved? The gold standard of proof for a molecular ruler is generally believed to be genetic replacement with ruler molecules of altered lengths, leading to corresponding predicted changes in filament lengths [5]. But this approach would not rule out a concentrative mechanism in which the M1M2M3 domain regulates tropomodulin capping at altered distances. Instead, the template function (the modular repeats) and the cap locator function (the M1M2M3 domain) must be physically dissociated. If the M1M2M3 domain were to be mislocalized, then tropomodulin capping would be increased in this new location and filament lengths would change correspondingly. The muscle sarcomere is a complex, highly ordered structure, but the molecules that make up the elements of this structure are dynamic and their length control cannot be explained by a simple ruler mechanism. A static ruler mechanism relying on precise stoichiometries cannot be used to determine sizes of dynamic polymers. This is because to allow for variations in filament length, cells would have to make multiple rulers of assorted lengths. Instead, cells have complex layers of regulation to allow for the reuse of their polymer building blocks in countless combinations and amounts to achieve many different outcomes. In the case of muscle, nebulin is one part of the combinatorial regulatory process that defines the precise filament lengths required for physiological functions. Determining the
molecular mechanisms by which muscle filament lengths are regulated will provide new paradigms to explain macromolecular size control. Dissecting the role of nebulin in regulating filament lengths is a good place to start.
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References 1. Fowler, V.M. (1997). Capping actin filament growth: tropomodulin in muscle and nonmuscle cells. Soc. Gen. Physiol. Ser. 52, 79–89. 2. Littlefield, R., and Fowler, V.M. (1998). Defining actin filament length in striated muscle: rulers and caps or dynamic stability? Annu. Rev. Cell Dev. Biol. 14, 487–525. 3. Trinick, J. (1994). Titin and nebulin: protein rulers in muscle? Trends Biochem. Sci. 19, 405–409. 4. Wang, K., and Wright, J. (1988). Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. J. Cell Biol. 107, 2199–2212. 5. Marshall, W.F. (2004). Cellular length control systems. Annu. Rev. Cell Dev. Biol. 20, 677–693. 6. Labeit, S., Gibson, T., Lakey, A., Leonard, K., Zeviani, M., Knight, P., Wardale, J., and Trinick, J. (1991). Evidence that nebulin is a protein-ruler in muscle thin filaments. FEBS Lett. 282, 313–316. 7. McElhinny, A.S., Kazmierski, S.T., Labeit, S., and Gregorio, C.C. (2003). Nebulin: the nebulous, multifunctional giant of striated muscle. Trends Cardiovasc. Med. 13, 195–201. 8. Kruger, M., Wright, J., and Wang, K. (1991). Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile. J. Cell Biol. 115, 97–107. 9. Labeit, S., and Kolmerer, B. (1995). The complete primary structure of human nebulin and its correlation to muscle structure. J. Mol. Biol. 248, 308–315. 10. Fischer, R.S., and Fowler, V.M. (2003). Tropomodulins: life at the slow end. Trends Cell Biol. 13, 593–601. 11. McElhinny, A.S., Kolmerer, B., Fowler, V.M., Labeit, S., and Gregorio, C.C. (2001). The N-terminal end of nebulin
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interacts with tropomodulin at the pointed ends of the thin filaments. J. Biol. Chem. 276, 583–592. McElhinny, A.S., Schwach, C., Valichnac, M., Mount-Patrick, S., and Gregorio, C.C. (2005). Nebulin regulates the assembly and lengths of the thin filaments in striated muscle. J. Cell Biol. 170, 947–957. Kazmierski, S.T., Antin, P.B., Witt, C.C., Huebner, N., McElhinny, A.S., Labeit, S., and Gregorio, C.C. (2003). The complete mouse nebulin gene sequence and the identification of cardiac nebulin. J. Mol. Biol. 328, 835–846. Littlefield, R., and Fowler, V.M. (2002). Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images. Biophys. J. 82, 2548–2564. Littlefield, R., Almenar-Queralt, A., and Fowler, V.M. (2001). Actin dynamics at pointed ends regulates thin filament length in striated muscle. Nat. Cell Biol. 3, 544–551. Sussman, M.A., Baque, S., Uhm, C.S., Daniels, M.P., Price, R.L., Simpson, D., Terracio, L., and Kedes, L. (1998). Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils. Circ. Res. 82, 94–105. Sussman, M.A., Welch, S., Cambon, N., Klevitsky, R., Hewett, T.E., Price, R., Witt, S.A., and Kimball, T.R. (1998). Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice. J. Clin. Invest. 101, 51–61. Gregorio, C.C., Weber, A., Bondad, M., Pennise, C.R., and Fowler, V.M. (1995). Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes. Nature 377, 83–86. Fritz-Six, K.L., Cox, P.R., Fischer, R.S., Xu, B., Gregorio, C.C., Zoghbi, H.Y., and Fowler, V.M. (2003). Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality. J. Cell Biol. 163, 1033–1044. Marshall, W. (2002). Size control in dynamic organelles. Trends Cell Biol. 12, 414–419.
Department of Cell Biology – CB163, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA. DOI: 10.1016/j.cub.2005.12.003
Developmental Biology: Micromanaging Muscle Growth Much remains to be learnt about the in vivo function of specific microRNAs. Recently, the conserved microRNA miR-1 has been found to be essential for Drosophila development. miR-1 mutants die during the rapid larval growth phase with severe muscle defects. Michael V. Taylor In the last few years, the widespread prevalence of microRNAs encoded in the genomes of organisms ranging from the nematode worm,
Caenorhabditis elegans, to humans has been discovered. MicroRNAs are short RNA molecules, around 22 nucleotides in length. As a group, they can control gene expression by two routes: promotion of mRNA
‘minimal’ model in olfactory research. References 1. Fishilevich, E., Domingos, A.I., Asahina, K., Naef, F., Vosshall, L.B., and Louis, M. (2005). Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila. Curr. Biol. 15, 2086-2096. 2. Buck, L., and Axel, R. (1991). A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175–187. 3. Clyne, P.J., Warr, C.G., Freeman, M.R., Lessing, D., Kim, J., and Carlson, J.R. (1999). A novel family of divergent seventransmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22, 327–338. 4. Vosshall, L.B., Amrein, H., Morozov, P.S., Rzhetsky, A., and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96, 725–736. 5. Ressler, K.J., Sullivan, S.L., and Buck, L.B. (1994). Information coding in the olfactory system: Evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245–1255. 6. Vassar, R., Chao, S.K., Sitcheran, R., Nunez, J.M., Vosshall, L.B., and Axel, R. (1994). Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981–991. 7. Gao, Q., Yuan, B., and Chess, A. (2000).
Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat. Neurosci. 3, 780–785. 8. Vosshall, L.B., Wong, A.M., and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102, 147–159. 9. Joerges, J., Küttner, A., Galizia, C.G., and Menzel, R. (1997). Representations of odours and odour mixtures visualized in the honeybee brain. Nature 387, 285–287. 10. Malnic, B., Hirono, J., Sato, T., and Buck, L.B. (1999). Combinatorial receptor codes for odors. Cell 96, 713–726. 11. Wang, J.W., Wong, A.M., Flores, J., Vosshall, L.B., and Axel, R. (2003). Twophoton calcium imaging reveals an odorevoked map of activity in the fly brain. Cell 112, 271–282. 12. Hildebrand, J.G., and Shepherd, G. (1997). Mechanisms of olfactory discrimination: converging evidence for common principles across phyla. Annu. Rev. Neurosci. 20, 595–631. 13. Robertson, H.M., Warr, C.G., and Carlson, J.R. (2003). Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 100, 14537–14542. 14. Couto, A., Alenius, M., and Dickson, B.J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15, 1535–1547. 15. Kreher, S.A., Kwon, A.Y., and Carlson,
Nebulin: Does It Measure up as a Ruler? A recent study has shown that the giant protein nebulin maintains the lengths of actin filaments in striated muscle cells. Although on the surface, nebulin looks like a molecular ruler, it may be playing a more complex role in regulating dynamics at the pointed end of actin filaments in striated muscle. Velia M. Fowler, Caroline R. McKeown and Robert S. Fischer In a striated muscle cell, the lengths of the sarcomeric actin filaments — the thin filaments — are precisely regulated, both in terms of their particular lengths, and in the variation of their length distributions [1,2]. Actin monomers assemble in vitro into filaments with an exponential length distribution, and they can organize into a wide variety of lengths in non-muscle cells, indicating that length controls are not intrinsic to actin filament polymers. Historically, a ‘molecular ruler’ mechanism has been the most attractive model for how thin filament lengths are determined in striated muscle. By definition, a
molecular ruler must meet a number of criteria: the ruler must be the precise length of the target filament, with the length of the ruler dictating the length of the filaments; the end of the ruler should bind to a terminator protein to prevent actin subunit addition or loss once the filament has polymerized to the length of the ruler protein; and the ruler must associate in a stoichiometric ratio with its target filament. In this mechanism, filament lengths are fixed precisely to the ruler length once they have polymerized. Thus, only filaments associated with rulers will be the length of the ruler, and filaments without a ruler will assume random and variable lengths (Figure 1A). The giant molecule nebulin has been postulated to be a molecular
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J.R. (2005). The molecular basis of odor coding in the Drosophila larva. Neuron 46, 445–456. Larsson, M.C., Domingos, A.I., Jones, W.D., Chiappe, M.E., Amrein, H., and Vosshall, L.B. (2004). Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43, 703–714. Hallem, E.A., Ho, M.G., and Carlson, J.R. (2004). The molecular basis of odor coding in the Drosophila antenna. Cell 117, 965–979. Goldman, A.L., van der Goes van Naters, W., Lessing, D., Warr, C.G., and Carlson, J.R. (2005). Coexpression of two functional odor receptors in one neuron. Neuron 45, 661–666. Ramaekers, A., Magnenat, E., Marin, E.C., Gendre, N., Jefferis, G.S.X.E., Luo, L., and Stocker, R.F. (2005). Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit. Curr. Biol. 15, 982–992. Wilson, R.I., Turner, G.C., and Laurent, G. (2004). Transformation of olfactory representations in the Drosophila antennal lobe. Science 303, 366–370.
Department of Biology, University of Fribourg, 10 rue du Musée, CH-1700 Fribourg, Switzerland. DOI: 10.1016/j.cub.2005.12.008
ruler that determines thin filament length in striated muscle [3–7]. Nebulin extends along the thin filament, with its amino terminus oriented near the pointed (free) end and its carboxyl terminus near the barbed end in the Z disc. A number of properties of nebulin appear to fulfill many of the a priori requirements for a molecular ruler. First, the molecular sizes of nebulin isoforms correlate with the lengths of thin filaments in the striated muscles in which the isoform is found [6,8]. Second, nebulin molecules are composed of a modular series of repeats corresponding to the repeats of the actin subunits of the thin filament, thus ‘measuring’ polymer length [6,9]. Third, a region in the unique amino-terminal domain of nebulin (M1M2M3) is located near the thin filament pointed end [9], and interacts with the actin pointed end capping protein, tropomodulin [10,11], thus potentially providing a mechanism to arrest filament elongation at precisely the length of the nebulin template [7] (Figure 1A). In a recent study, Gregorio and colleagues [12] have shown for the first time that nebulin regulates thin filament length. Using RNA
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Figure 1. Models for B Cap locator A Ruler regulation of thin filament Z Z lengths in striated muscle. (A) The ruler mechanism requires that actin subunits polymerize into filaments alongside the nebulin ruler. Actin assembly is terminated at the length of the ruler by tropomodulin (Tmod), which binds tightly to the actin pointed end and to the M1M2M3 domain of nebulin. Regulation of filament length is stoichiometric, requiring each filament to be associated with a nebulin ruler and tightly capped at its pointed end by tropomodNebulin Actin ulin. When filaments are M1M2M3 domain Tmod not associated with rulers, Current Biology for example when nebulin levels are reduced or absent, then filaments are not length regulated and randomly assume both shorter and longer lengths (middle and bottom). Filament lengths are not influenced by increases in the concentration of tropomodulin nor by rates of actin monomer association and dissociation at filament ends. (B) The cap locator mechanism depends on localized regulation of tropomodulin capping and actin dynamics. Dynamic association of tropomodulin with the M1M2M3 domain of nebulin generates an increased local concentration of tropomodulin at a distance from the Z disc (Z) determined by the length of the nebulin. Increased tropomodulin capping of pointed ends in this location competes for actin subunit addition and controls lengths. Lengths do not depend on precise stoichiometric ratios of filaments to rulers, and are sensitive to both tropomodulin concentration and actin dynamics. When nebulin levels are reduced or absent, the local tropomodulin concentration falls, allowing actin subunit addition and graded and uniform elongation of filaments. The precision of length regulation (variability) depends on the relative local concentration of tropomodulin, which depends on nebulin levels and on the affinity of tropomodulin for the M1M2M3 domain of nebulin [11]. Decreasing amount of nebulin
interference technology, nebulin levels were knocked down in cultured rat cardiac myocytes and sarcomere organization and thin filament lengths were evaluated by fluorescence microscopy in both fixed and living cells. Strikingly, after reduction of nebulin levels, actin and tropomyosin were found to extend across the gap in the middle of the sarcomere, indicating that thin filaments had elongated from their pointed ends. No changes were observed in Z disc distances or in the striated organization of titin or thick filaments, demonstrating that sarcomeres were not hypercontracted. Further, tropomodulin was no longer associated with sarcomeres, showing that it had dissociated from pointed ends of the aberrantly elongated thin filaments, as expected if the capping of thin filaments by tropomodulin depends on nebulin. The observed dependence of tropomodulin localization and thin filament length on nebulin levels are both consistent with the idea that nebulin could be a thin filament molecular ruler. Certain results in this study [12], however, lead to the conclusion that nebulin does not regulate length by a simple physical ruler mechanism. At intermediate times, sarcomeres with partially reduced staining for nebulin displayed narrower gaps in actin staining in the middle of the sarcomere. Line scans of the fluorescence intensity across the sarcomeres showed that partial reductions in nebulin led to uniform elongation of thin filaments in individual sarcomeres by up to 30%. This is completely inconsistent with a molecular ruler function for nebulin. If nebulin were a true ruler, the filaments without nebulin would have been expected to assume random lengths, while filaments with nebulin should have remained at their original length (Figure 1A, middle). Instead, fewer nebulins led to graded and uniform increases in filament length (Figure 1B, middle). Comparison of nebulin levels and thin filament lengths in intact muscles also reveals difficulties with a molecular ruler mechanism. While nebulin is present in skeletal
muscle at stoichiometric levels of two per filament [3,7], it is considerably less abundant in cardiac muscle [4,13] where the lengths of thin filaments are still regulated [2,14]. Moreover, myofibrils isolated from some skeletal muscle types have thin filament lengths that vary by about 100 nm [14], despite the predominance of one nebulin isoform in each muscle type [8]. Significantly, a key feature of thin filament length control that is not explained by a molecular ruler mechanism is the role of actin dynamics. Lengths of muscle thin filaments are controlled by regulation of actin assembly at the pointed, but not barbed, filament ends. The pointed ends of actin filaments in muscle are transiently capped by tropomodulin, allowing for continuous actin subunit exchange [15]. An increase in the tropomodulin level reduces actin incorporation at pointed ends and leads to uniform shortening of thin filaments [15–17]. Conversely, a
decrease in the tropomodulin level or inhibition of tropomodulin’s actin-capping activity results in thin filament elongation [16,18,19]. While these experiments demonstrate that regulation of actin assembly dynamics by tropomodulin controls filament lengths, determination of specific lengths requires that rates of assembly must be lengthdependent [2,5,20]. We propose that nebulin may be a ‘cap locator’ that provides a position-dependent regulation of tropomodulin capping to determine thin filament lengths (Figure 1B). In the simplest mechanism, dynamic interaction of tropomodulin with the aminoterminal M1M2M3 region of nebulin near the pointed end could serve to create a localized concentration of tropomodulin, at a distance from the Z disc determined by the length of nebulin, as suggested previously by Marshall [5]. In this mechanism, nebulin molecules
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need not be stoichiometric with each thin filament, nor does nebulin regulate filament lengths in a binary, all-or-nothing fashion. As the numbers of nebulins per sarcomere are decreased, the local concentration of tropomodulin is diminished, leading to lower effective capping activity, increased actin assembly and uniformly longer thin filaments (Figure 1B). This model is also consistent with the dependence of thin filament length on tropomodulin concentrations [15–17]. How can the question of whether nebulin acts as a molecular ruler or a cap locator be resolved? The gold standard of proof for a molecular ruler is generally believed to be genetic replacement with ruler molecules of altered lengths, leading to corresponding predicted changes in filament lengths [5]. But this approach would not rule out a concentrative mechanism in which the M1M2M3 domain regulates tropomodulin capping at altered distances. Instead, the template function (the modular repeats) and the cap locator function (the M1M2M3 domain) must be physically dissociated. If the M1M2M3 domain were to be mislocalized, then tropomodulin capping would be increased in this new location and filament lengths would change correspondingly. The muscle sarcomere is a complex, highly ordered structure, but the molecules that make up the elements of this structure are dynamic and their length control cannot be explained by a simple ruler mechanism. A static ruler mechanism relying on precise stoichiometries cannot be used to determine sizes of dynamic polymers. This is because to allow for variations in filament length, cells would have to make multiple rulers of assorted lengths. Instead, cells have complex layers of regulation to allow for the reuse of their polymer building blocks in countless combinations and amounts to achieve many different outcomes. In the case of muscle, nebulin is one part of the combinatorial regulatory process that defines the precise filament lengths required for physiological functions. Determining the
molecular mechanisms by which muscle filament lengths are regulated will provide new paradigms to explain macromolecular size control. Dissecting the role of nebulin in regulating filament lengths is a good place to start.
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References 1. Fowler, V.M. (1997). Capping actin filament growth: tropomodulin in muscle and nonmuscle cells. Soc. Gen. Physiol. Ser. 52, 79–89. 2. Littlefield, R., and Fowler, V.M. (1998). Defining actin filament length in striated muscle: rulers and caps or dynamic stability? Annu. Rev. Cell Dev. Biol. 14, 487–525. 3. Trinick, J. (1994). Titin and nebulin: protein rulers in muscle? Trends Biochem. Sci. 19, 405–409. 4. Wang, K., and Wright, J. (1988). Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. J. Cell Biol. 107, 2199–2212. 5. Marshall, W.F. (2004). Cellular length control systems. Annu. Rev. Cell Dev. Biol. 20, 677–693. 6. Labeit, S., Gibson, T., Lakey, A., Leonard, K., Zeviani, M., Knight, P., Wardale, J., and Trinick, J. (1991). Evidence that nebulin is a protein-ruler in muscle thin filaments. FEBS Lett. 282, 313–316. 7. McElhinny, A.S., Kazmierski, S.T., Labeit, S., and Gregorio, C.C. (2003). Nebulin: the nebulous, multifunctional giant of striated muscle. Trends Cardiovasc. Med. 13, 195–201. 8. Kruger, M., Wright, J., and Wang, K. (1991). Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile. J. Cell Biol. 115, 97–107. 9. Labeit, S., and Kolmerer, B. (1995). The complete primary structure of human nebulin and its correlation to muscle structure. J. Mol. Biol. 248, 308–315. 10. Fischer, R.S., and Fowler, V.M. (2003). Tropomodulins: life at the slow end. Trends Cell Biol. 13, 593–601. 11. McElhinny, A.S., Kolmerer, B., Fowler, V.M., Labeit, S., and Gregorio, C.C. (2001). The N-terminal end of nebulin
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interacts with tropomodulin at the pointed ends of the thin filaments. J. Biol. Chem. 276, 583–592. McElhinny, A.S., Schwach, C., Valichnac, M., Mount-Patrick, S., and Gregorio, C.C. (2005). Nebulin regulates the assembly and lengths of the thin filaments in striated muscle. J. Cell Biol. 170, 947–957. Kazmierski, S.T., Antin, P.B., Witt, C.C., Huebner, N., McElhinny, A.S., Labeit, S., and Gregorio, C.C. (2003). The complete mouse nebulin gene sequence and the identification of cardiac nebulin. J. Mol. Biol. 328, 835–846. Littlefield, R., and Fowler, V.M. (2002). Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images. Biophys. J. 82, 2548–2564. Littlefield, R., Almenar-Queralt, A., and Fowler, V.M. (2001). Actin dynamics at pointed ends regulates thin filament length in striated muscle. Nat. Cell Biol. 3, 544–551. Sussman, M.A., Baque, S., Uhm, C.S., Daniels, M.P., Price, R.L., Simpson, D., Terracio, L., and Kedes, L. (1998). Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils. Circ. Res. 82, 94–105. Sussman, M.A., Welch, S., Cambon, N., Klevitsky, R., Hewett, T.E., Price, R., Witt, S.A., and Kimball, T.R. (1998). Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice. J. Clin. Invest. 101, 51–61. Gregorio, C.C., Weber, A., Bondad, M., Pennise, C.R., and Fowler, V.M. (1995). Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes. Nature 377, 83–86. Fritz-Six, K.L., Cox, P.R., Fischer, R.S., Xu, B., Gregorio, C.C., Zoghbi, H.Y., and Fowler, V.M. (2003). Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality. J. Cell Biol. 163, 1033–1044. Marshall, W. (2002). Size control in dynamic organelles. Trends Cell Biol. 12, 414–419.
Department of Cell Biology – CB163, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA. DOI: 10.1016/j.cub.2005.12.003
Developmental Biology: Micromanaging Muscle Growth Much remains to be learnt about the in vivo function of specific microRNAs. Recently, the conserved microRNA miR-1 has been found to be essential for Drosophila development. miR-1 mutants die during the rapid larval growth phase with severe muscle defects. Michael V. Taylor In the last few years, the widespread prevalence of microRNAs encoded in the genomes of organisms ranging from the nematode worm,
Caenorhabditis elegans, to humans has been discovered. MicroRNAs are short RNA molecules, around 22 nucleotides in length. As a group, they can control gene expression by two routes: promotion of mRNA