QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 March 2005
doi: 10.1242/jcs.02281

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qiu, F. Articles by Bullard, B. Search for Related Content PubMed PubMed Citation Articles by Qiu, F. Articles by Bullard, B. Social Bookmarking                         What's this?

Myofilin, a protein in the thick filaments of insect muscle

Feng Qiu^*, Sigrun Brendel, Paulo M. F. Cunha, Nagore Astola, Bauzhen Song, Eileen E. M. Furlong, Kevin R. Leonard and Belinda Bullard

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (47K): [in a new window]   Fig. 2. Comparison of the sequences of myofilin and the sequences of flightin in different insects. (A) Alignment of myofilin amino acid sequences in Drosophila melanogaster (isoforms DmMf5 and DmMf2), Anopheles gambiae (AgMf2) and Lethocerus indicus (LiMf2). Drosophila and Lethocerus myofilin sequences were obtained as described in the Materials and Methods. The homologous sequence in Anopheles is from the EST database (BM64635). Caenorhabditis elegans ß-filagenin sequence is shown below the myofilin sequences (Liu et al., 1998). Stars below the sequence are regions in DmMf5 predicted to be helical. (B) Alignment of flightin sequences in D. melanogaster (DmFln) and L. indicus (LiFln). No flightin sequence was found in the Anopheles genome. In both A and B, residues that are identical in Lethocerus and Drosophila or Anopheles or C. elegans, or all four, are shaded dark grey; other identical residues are shaded light grey. This illustrates the extent of homology between the sequences of the hemipteran, Lethocerus, and the two dipterans, Drosophila and Anopheles, as well as that between the sequences of individual insect species; C. elegans ß-filagenin is included for comparison with a nematode.

View larger version (17K): [in a new window]   Fig. 1. Alternative splicing of the D. melanogaster myofilin gene. (A) The top line shows the exon-intron organisation of the myofilin gene, which contains nine exons. The start codon (green) is found in exon 2, stop codons (red) are found in exons 5, 8 and 9. Exons 5, 6, and 7 have internal splice sites, alternative versions are indicated by a (upper) and b (lower). (B) Schematic representation of the structure of mRNAs encoded by the myofilin gene. The exons present in the different isoforms DmMf1 to DmMf5 are shown.

View larger version (56K): [in a new window]   Fig. 3. Isoforms of myofilin in Drosophila and myosin binding to myofilin. (A) An immunoblot of thoraces from wild-type Drosophila (WT) and mutant flies lacking thick filaments in the IFM (Mhc7), and isolated wild-type flight muscle (IFM) was incubated with anti-myofilin antiserum. There are five isoforms of myofilin in muscles of the thorax. Mhc7 lacks the 20 kDa isoform, which is the sole isoform in IFM. The strip on the right shows a blot of thoraces from wild-type flies incubated with anti-flightin. (B) Overlay assay of myosin binding to immunoblots of Lethocerus IFM. Lane1 was incubated in myosin antibody (MAC 147); lane 2 was incubated in buffer containing Lethocerus myosin (0.1 mg/ml) and then in myosin antibody. Myosin bound to myofilin and to other proteins (paramyosin, Pm; mini-paramyosin, mPm; and actin). Myofilin (30 kDa) has an anomalously low migration rate on SDS-PAGE gels. Myosin also bound strongly to the two myosin light chains (Mlc1 and Mlc2); there are three variants of Mlc2, which differ in the extent of phosphorylation and have slightly different mobilities. Myosin did not bind to flightin (23 kDa). Mhc, myosin heavy chain.

View larger version (112K): [in a new window]   Fig. 4. The position of myofilin in Drosophila muscles. (A) Thin, slightly oblique cryosection of Drosophila IFM labelled with anti-myofilin and Protein A-gold. The fibre is slightly stretched to increase the length of the I-band. Gold particles are in diagonal lines across the sarcomere; this results from exposure of epitopes by the knife cutting through the regular thick filament lattice (Ferguson et al., 1994). The positions of H-zone (H), A-band (A), I-band (I), Z-band (Z) are shown. Horizontal lines mark the extent of the A-band. Beneath the micrograph is a histogram showing the distribution of gold particles and the corresponding density profile across the sarcomere. Gold particles extend to the edges of the A-band and H-zone. (B) Cryosection of non-flight muscle labelled with anti-myofilin and Protein A-gold. In this case the myofibrils are narrower and the sarcomeres longer, so that the H-zones are not visible in this micrograph. Labelling is less regular, but again extends to the end of the A-band. Bar, 200 nm.

View larger version (66K): [in a new window]   Fig. 5. Distribution of myofilin in Drosophila IFM thick filaments. Cryosections were labelled with anti-myofilin and Protein A-gold. Panels show selected runs of gold particles following the line of the thick filaments. The mean value for the smallest regular spacing between gold particles was at 30±5 nm (mean±s.d., n=42, taken from three images). Bar, 120 nm.

View larger version (77K): [in a new window]   Fig. 6. Expression of myofilin RNA and protein in the Drosophila embryo. (A) Stage 15-17 embryo hybridised with a digoxigenin-labelled myofilin RNA probe. (B) Higher magnification of the same embryo showing labelling of somatic muscles, but not heart or visceral muscles. (C) Stage 15-17 embryo labelled with anti-myofilin antiserum, showing specific immunostaining of somatic muscles. (D) Higher magnification of the same embryo. Labelling of individual muscles can be clearly seen at this embryonic stage. Embryos are seen in lateral view with anterior to the left.

View larger version (46K): [in a new window]   Fig. 7. Northern blot analysis of the expression of myofilin in Drosophila. Lanes 1 to 7 are stages of embryonic development as follows: lane 1, 0-1 hours (stages 1-3); lane 2, 1-3 hours (stages 1-6); lane 3, 3-6 hours (stages 7-11); lane 4, 6-9 hours (stages 11-13); lane 5, 9-12 hours (stages 13-15); lane 6, 12-15 hours (stages 15-16); lane 7, 15-18 hours (stages 16-17). Lanes L1, L2 and L3 are first, second and third instar larvae. Lanes F and M are female and male adults. Expression of myofilin is detected as a strong band corresponding to a predicted size of 1.3 kb, starting at 15-18 hours of development or stage 16 and present in all larval stages and in the adults. A faint band is also seen that could correspond to the splice variant of predicted size 0.8 kb (arrow). This band is not detected at any stage of embryogenesis but is present throughout larval life and in the adults. Rp49 was hybridised to the same blot as the myofilin probe (lower image) as a loading control.

View larger version (33K): [in a new window]   Fig. 8. A model for the arrangement of myofilin and flightin in the IFM thick filament. (A) Cross-sectional top view of half a thick filament showing the myosin sub-filaments (adapted from Beinbrech et al., 1988). In the full thick filament there would be 12 sub-filaments, here we show only numbers 1-3 and 10-12. Myofilin molecules are shown in grey on the inside of the thick filament. Flightin molecules are open symbols on the outside of the filament. (B) A side view of the thick filament lattice, again based on Beinbrech's model. Each sub-filament has myosin molecules with alternating `outer' heads (black ellipses pointing to the left) and `inner' heads pointing to the right. The myofilin molecules only bind to the rod region of the `inner' myosin molecules, with the result that they are arranged at an interval of 29 nm, as shown; this is twice the 14.5 nm spacing of the myosin heads in the filament. Myofilin is placed at an arbitrary position on the myosin rod. Flightin molecules are bound to `outer' myosin molecules about two-thirds of the way along the rod. In order to simplify the representation of the model, only one head of myosin dimers is shown, and the sub-filaments are drawn parallel to the axis of the thick filament, although evidence from X-ray diffraction suggests that they are inclined at a shallow angle to the filament (Wray, 1979b).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter