Number and spatial distribution of nuclei in the muscle fibres of normal mice studied in vivo - PubMed (original) (raw)

Number and spatial distribution of nuclei in the muscle fibres of normal mice studied in vivo

J C Bruusgaard et al. J Physiol. 2003.

Abstract

We present here a new technique with which to visualize nuclei in living muscle fibres in the intact animal, involving injection of labelled DNA into single cells. This approach allowed us to determine the position of all of nuclei within a sarcolemma without labelling satellite cells. In contrast to what has been reported in tissue culture, we found that the nuclei were immobile, even when observed over several days. Nucleic density was uniform along the fibre except for the endplate and some myotendinous junctions, where the density was higher. The perijunctional region had the same number of nuclei as the rest of the fibre. In the extensor digitorum longus (EDL) muscle, the extrajunctional nuclei were elongated and precisely aligned to the long axis of the fibre. In the soleus, the nuclei were rounder and not well aligned. When comparing small and large fibres in the soleus, the number of nuclei varied approximately in proportion to cytoplasmic volume, while in the EDL the number was proportional to surface area. Statistical analysis revealed that the nuclei were not randomly distributed in either the EDL or the soleus. For each fibre, actual distributions were compared with computer simulations in which nuclei were assumed to repel each other, which optimizes the distribution of nuclei with respect to minimizing transport distances. The simulated patterns were regular, with clear row-like structures when the density of nuclei was low. The non-random and often row-like distribution of nuclei observed in muscle fibres may thus reflect regulatory mechanisms whereby nuclei repel each other in order to minimize transport distances.

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Figures

Figure 3

Figure 3. The position of nuclei observed with an interval of 24 h to 4 days

A and B are micrographs of two different fibres observed with an interval of 24 h and 4 days (4 d), respectively. Corresponding nuclei are connected by lines. Scale bar = 50 μm. C, surface foldout of a third fibre; corresponding nuclei are given the same number designation. Note the constancy of nuclear positions. Grey symbols indicate synaptic nuclei.

Figure 1

Figure 1. Density of nuclei along the length of the fibre, from the endplate (0 μm) to the distal tendon

Each point represents the mean ±

s.e.m

. of three fibres taken from the EDL and soleus (SOL).

Figure 2

Figure 2. Three- and two-dimensional reconstruction of nucleic positions in a fibre segment

A, averaged micrograph of 17 focal planes of the segment. Scale bar = 50 μm. B, reconstruction seen from the same angle as in A, and from the end (× 4 higher magnification). An oval cylindrical surface (dotted) was fitted to the data and the positions of the nuclei were projected onto this virtual surface. C, for further analysis, the surface was ‘cut open’ and folded out so as to give the positions of the nuclei in the two-dimensional cell. The borders of the foldout, roughly corresponding to the sarcolemma, are indicated by the dotted line.

Figure 4

Figure 4. The shape and orientation of single nuclei in soleus and EDL

A, micrographs of 30 nuclei from both the EDL and soleus (each square is 20 μm wide). B, eccentricity indices (see Methods) of the same nuclei as depicted in A, and of synaptic (end plate, EP) nuclei in the EDL. C, orientation of the long axis of the non-synaptic nuclei relative to the long axis of the fibre for the EDL and soleus, and for EP nuclei from an EDL fibre.

Figure 5

Figure 5. Endplate nuclei and nuclei in the perisynaptic region

Micrograph (A, scale bar 20 μm) and foldout (B) of a fibre segment in the endplate region. In A, acetylcholine receptors labelled with α-bungarotoxin (red) and nuclei labelled with fluorescein-conjugated DNA (green) is shown. C, the nuclear position relative to the endplate on foldouts from nine fibres superimposed. No perisynaptic specialization was apparent.

Figure 6

Figure 6. Nuclei at the myotendinous region

Micrographs of examples of fibre segments at the distal end of the fibre, showing the myotendinous junction without specialization (A) and with an increased density of nuclei (B) as observed in five out of 19 fibres. Scale bar = 50 μm.

Figure 7

Figure 7. Cell size and nuclear domains

A, average nuclear cytoplasmic domain volumes; B, average cell surface domain areas; and C, nuclear density of EDL and soleus fibres with different cross-sectional areas. In C, the theoretical nuclear density with a constant nuclear cytoplasmic domain volume is denoted by a straight line and that with a constant nuclear sarcolemmal domain area by a curved line. The curves were constructed by least-squares linear and non-linear regression, assuming an unchanged form of the fibre. The linear regression coefficients were statistically different (*) or not different (n.s.) from 0.

Figure 8

Figure 8. Distribution patterns of nuclei in one fibre segment as observed and as obtained by positioning the same nuclei by computer simulations

A, averaged micrograph of 17 focal planes of the segment. Scale bar = 50 μm. B, observed nuclear positions on the surface foldout. C, a computer simulation where the same number of nuclei were positioned randomly on the same surface. D, a computer simulation where the nuclei were assumed to repel each other in order to form patterns that should be near optimal in avoiding long transport distances along the surface of the fibre. E, a computer simulation where nuclei were assumed to repel each other and distances were measured directly through the cytosol. F, same fibre as in E but with added normally distributed noise (

s.d.

equal to one-third of the average distances in E).

Figure 9

Figure 9. Distribution of nearest neighbour distances for nuclei as observed or by random positioning

Data are the mean of 19 EDL fibres and 14 soleus fibres. The distances are given relative to the optimal distance given by the computer simulations (direct and along the surface) calculated for each fibre. The optimal (maximal) distance was set to 1.

Figure 10

Figure 10. Nuclear row formation

The distribution of the nuclei in two fibre segments seen from the end. A, the best example of nuclear preference for selected sectors of the circumference (P = 0.0006, Monte Carlo simulation). B, in this fibre the nuclei had a more random distribution around the circumference (not significant, Monte Carlo simulation).

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