(see also "Several Insights into muscle contraction mechanism " part 1 and 2)



About the nature of the hypothetical ultra-thin N filament




The hypothetical ultra-thin N filament are imagined here as a thread that connect the so-called "free end" of a thin filament to the Z line at the opposite edge of the sarcomere.




      There are reasons to consider that the hypothetical ultra-thin N filaments are made up of nebulin:


 a) nebulin is a N2 line associated protein  (Wang & Williamson, 1980, Identification of an N2 line protein of striated muscle, Proc.Natl Acad Sci. USA, 77, 3254 - 3258



b) N2 line divide the distance between the pointed ends of thin filaments and the Z line at the opposite end of the sarcomere  in two segments whose ratio is constant, no matter what the length of sarcomere (i.e., the N2 lines behaves as a homothetic center for  this distance) (Dan Eremia, 1985)

c) nebulin (similar to titin) is an elastic protein polymer made up of serially connected monomers

d) nebulin monomers may exist either in a folded or in an unfolded configuration.


        Nebulin is a giant (600-900 kDa) modular sarcomeric protein which still remains one of the most "nebulous" components of the striated muscle. Human nebulin has a molecular weight of 773 kDa and consists of 185 tandem copies of an 35-amino acid residue module.

Each module has a conserved motif thought to interact with a single actin monomer. Modules 9-162 are organized into sets of seven-module super-repeats, which contain a second conserved motif that matches the periodicity of (and is thought to organize) the tropomyosin/troponin complexes (Jin and Wang, 1991; Pfuhl et al., 1994; Labeit and Kolmerer, 1995; Wang et al., 1996).

       The C terminus of nebulin extends into the Z-disc, in which it interacts with α-actinin, myopalladin and the intermediate filament protein desmin (Nave et al., 1990; Bang et al., 2001, 2002).

Immuno-fluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with tropomodulin (Tmod) at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod, as demonstrated by blot overlay, bead binding and solid phase binding assays. http://www.jbc.org/content/276/1/583.full

These data may support the hypothesis according to that nebulin molecule spans the entire length of the thin filament.

Currently, nebulin molecule are  supposed to act as a "ruler" to specify the thin filaments lengths.



However, the same immuno-fluorescence microscopy data may also support another hypothesis, according to that the nebulin molecule emerges from the pointed ends of thin filament toward the Z line at the opposite end of the sarcomere (instead of laying along the thin filament). Interestingly, even in this situation the N-terminal end of nebulin still colocalizes with Tmod and its C- terminal is still inserted in the Z line.




Amazingly enough, both hypothesis may equally be true. In fact (according to the immuno-electron microscopy data) there are two nebulin molecules ("Two nebulin molecules extend along the length of each 1-μm long actin filament to form a thin filament") extending along the each thin filament. (Labeit and Kolmerer 1995, The complete primary structure of human nebulin and its correlation with muscle structure, J. Mol. Biol.248: 308-315). http://www.ncbi.nlm.nih.gov/pubmed/7739042 





In 2009, Castillo, Nowak, Littlefield, Fowler and Littlefield ("A nebulin ruler does not dictate thin filament lengths", Biophys J. 2009 96:1856-65) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717268 found (in seven different rabbit skeletal muscles), using fluorescent microscopy and quantitative image analysis, that nebulin extended only 1.01-1.03 micrometer from the Z-line, but Tmod were localized 1.13-1.31 micrometer from the Z-line. Because nebulin does not extend to the thin filament pointed ends, it can neither target Tmod capping nor specify thin filament lengths.






     Pappas, Krieg, and Gregorio ("Nebulin regulates actin filament lengths by a stabilization mechanism" 2010 JCB 189: 859-870) constructed a unique, small version of nebulin (mini-nebulin) and replaced endogenous nebulin with mini-nebulin in skeletal myocytes. They observed that thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function.



     However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were maintained at lengths longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with (?!). Obviously, admitting that the pointed end of thin filament is anchored to the Z line at the opposite edge of the sarcomere by another nebulin molecule (i.e., our hypothetical ultra-thin N filament), this paradox may be easily explained.



     At the early stages of myofibril growth, the central part of titin may play a crucial role as a molecular scaffold for organizing the thick filament, the two peripheral segments becoming the Z-thick filament structures described by Ullrick et al. (Ullrick, Toselli, Chase, Dase 1977, J. Ultrastructure Res. 60, 263).

Similarly, if one of the two serially interconnected nebulin molecules may act as a scaffold for organizing thin filament http://jcb.rupress.org/content/170/6/947.full, the other nebulin molecule may remain not associated to any other structure, becoming our hypothetical ultra-thin N filament.


Apparently, nebulin is absent in cardiac muscle. Some new data show that nebulin is, however, expressed in cardiac muscle, mainly in atrial cardio-myocytes and, in a low percentage, of ventricular cardio-myocytes (Fock and Hinssen, 2002; Kazmierski et al., 2003; Bang et al., 2006).

A related protein, made up by 23 nebulin-like modules (named "nebulette" by Wang and Moncman in 1995, because of its smaller molecular weight, i.e. 107 kD compared with 600-900 kD) may provide similar functions.

Nebulette is (generally) predicted to extend from the  Z-line along the thin filament, but not able to reach its pointed end:



The role of nebulette are especially intriguing, given the growing number of mutations in this giant molecule that are associated with human cardio-myopathies.

Nebulette is, obviously, too short to act as a "ruler" for the thin filaments lengths in cardiac muscle. Let alone its extreme shortness, nebulette modules do not display a super-repeat pattern, as nebulin does (Moncman and Wang, 1999, Cell Motil. Cytoskeleton 44: 1-22). So, excepting the cardio-myocytes where nebulin is present (according to some recent data), in the remaining cardiac cells, the thin filaments may have not a nebulin ruller.

Interestingly enough, thin filament lengths are more precisely determined in the skeletal muscle sarcomeres, as compared with cardiac muscle, possibly because nebulette obviously does not act as a "ruler" for the thin filaments lengths. Thin filaments in rabbit cardiac muscle vary in length from 0.8 micrometer to about 1.3 micrometer (30% variation), while the distribution of thin filament lengths in skeletal muscle (1.11 +/- 0.03 micrometer) is significantly narrower (only 3% variation).

However, like nebulin molecule in skeletal musclenebulette molecule may also emerge from pointed ends of the thin filament toward the Z line at the opposite end of the sarcomere where it inserts its C- terminal. 




Non-activated preparations of cardiac muscle have long been known to be much stiffer than those of skeletal muscle.

According to Linke et al. (Linke, Ivemeyer, Labeit, Hinssen, Ruegg and Gautel "Actin-titin interaction in cardiac myofibrils: probing a physiological role" Biophysical Journal 1997, 73, 905-919) http://ukpmc.ac.uk/classic/pagerender.cgi?artid=486866&pageindex=1, the high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin. It was experimentally proven that actin extraction from isolated cardiac myofibrils (by a calcium-independent gelsolin fragment) decreases  by nearly 60% this stiffness.

      It is very interesting fact that, in contrast with the situation found in cardiac myofibrils, where stiffness decreases with actin extraction, in skeletal muscle (e.g. rabit psoas fibers), after thin filament extraction, passive force remains almost constant (Funatsu et al. 1990) or even increases significantly (Granzier and Wang, 1993).

      A pair of thick and thin filaments, together with the giant macromolecules titin and nebulin that they are interacting with are shown (in accord with the current opinion) in the following picture:

     After treatment with gelsoline and thin filament extraction, this picture will be modified:


       Is difficult to understand how actin extraction could possibly produce an increased stiffnes. One possibility would be that, in skeletal muscle, thin filaments may be able to resist extraction (Sanger et al. 1987, Huckriede et al. 1988). Gonsior and Hinssen (1995) showed that this resistance is, probably, conferred by nebulin. Another possibility is that actin extraction promotes interaction between the stiff nebulin and titin, so titin filament may become less compliant than in the intact sarcomere, so that at the given sarcomere length, myofibril stiffness is enhanced (cf. Granzier and Wang, 1993).

     Obviously, admitting that the pointed end of thin filament is anchored to the Z line at the opposite edge of the sarcomere by another nebulin molecule (our hypothetical ultra-thin N filament), this situation may be easily explained. The nebulin shown in the right part of the picture is no more under to constraint of thin filament and may behave similarly with the nebulin shown in the left part of the picture, i.e. some of its domains may fold in order to raise the passive force of the myofibril. It is already known that titin molecule (and very probable nebulin molecule) behaves as an entropic spring in which unfolding of the globular domains occurs at high force during stretch and refolding at low force during release.


     In the cardiac myofibrils, actin extraction by gelsolin should leave titin as the sole determinant of myofibril stiffness. It is unclear whether nebulette (in cardiac myofibril), similarly to nebulin in skeletal muscle, could act to prevent from extraction by gelsolin the actin existing near the Z-disk.

     In conclusion, the presence of nebulin in skeletal but not in cardiac muscle might explain why gelsolin treatment has different effects on stiffness in the two muscle types.

    One may think that all the up mentioned authors were very close to say that an explanation of this high stiffness may be simply the shortness of a connection between the "free ends" of the thin filaments and the Z line in the opposite edge of the sarcomere (which connection we consider here to be made of by the giant macromolecule nebulette substantially  shorter than nebulin). The authors acknowledged in this way the fact that thin filaments are, somehow, anchored to the opposite site of the sarcomere.


The shortness of the cardiac muscle N ultra-thin filament (only 23 modules as compared with the 153 modules of  the N filament in skeletal muscle) may have important repercussions in the cardiac muscle function: