Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibres
Gordon J. Lutz*,
Shannon N. Bremner,
Michael J. Bade and
Richard L. Lieber
Departments of Orthopaedics and Bioengineering, Biomedical Sciences Graduate Group, University of California, Veterans Affairs Medical Center and Veterans Medical Research Foundation, 3350 La Jolla Village Drive, San Diego, CA 92161, USA
View larger version (43K):
[in a new window]
|
Fig. 1. Identification of MLCs in single fibres representing the full range of fibre types in R. pipiens. (A) MHC isoform composition of single fibres was determined by SDS-PAGE. Shown are MHC bands from single fibres representing each of the major and intermediate fibre types (types 1, 1-2, 2, 2-3, 3 and tonic T). All fibres were obtained from one frog. Fibres were typed by comparison with the known positions of MHC isoforms from a standard whole muscle homogenate (anterior tibialis muscle). Fibre types, based on MHC content, are indicated at the bottom of each gel lane. (B) Analysis of MLCs in each of the MHC-typed fibres shown in A (fibre types labeled at bottom). (Left) SDS-PAGE (silver stained) of each fibre. Gel markers in leftmost lane have molecular masses (from top to bottom) of 45, 31, 21.5 and 14.5 kDa. (Middle) Western blots of the gel on left using monoclonal MLC antibodies T14, MF5 and F310. (Right) Schematic representation based on western blots of the MLC composition of the fibre types from this frog and 23 other frogs with a similar MLC composition.
|
|
View larger version (28K):
[in a new window]
|
Fig. 2. In some frogs, a novel MLC, MLC1x, is expressed in all fibre types. (Left) SDS-PAGE (silver stained) of single fibres representing most of the different fibre types. All fibres (fibre types labeled at bottom of gel lanes) were obtained from two frogs and were typed by their MHC banding pattern by SDS-PAGE, as in Fig. 1 (not shown). (Middle) Western blots of the gel on left using monoclonal MLC antibodies T14, MF5 and F310. (Right) Schematic representation based on western analysis of the MLC composition of the fibre types from this frog and six other frogs with similar MLC composition. A unique type of MLC1, designated as MLC1x, was expressed in all fibre types, producing a strikingly different expression pattern from those seen in fibres examined in Fig. 1.
|
|
View larger version (36K):
[in a new window]
|
Fig. 3. Identification of troponin I (TnI) and troponin C (TnC) isoforms in the various R. pipiens fibre types. (Left) SDS-PAGE (silver stained) banding pattern of single skinned fibres representing most of the different fibre types. All fibres (fibre types labeled at bottom of gel lanes) were obtained from one frog and were typed by MHC banding pattern after SDS-PAGE as in Fig. 1 (not shown). (Right) Western blots of the gel on left using anti-TnI and anti-TnC antibodies. Anti-TnI labeled one isoform in fast twitch fibres (TnIf) and a unique isoform in tonic fibres (TnIT). Anti-TnC labeled an isoform in tonic fibres (TnCT) that was not expressed in any other fibre type, but did not react with the TnC isoform present in fast twitch fibres (TnCf). The position of TnCf was deduced from indirect evidence (see Results for details). The lightly stained band below MLC2f in twitch fibres is an unidentified contaminant that is not typically present (see Figs 1, 2 and 4). Note, this frog did not contain the MLC1x isoform in any of its fibres.
|
|
View larger version (44K):
[in a new window]
|
Fig. 4. Summary of the expression pattern of MLC and troponin isoforms in fast twitch and tonic fibres. This figure combines the information from Figs 1 and 3 to indicate the positions of each of the identified myofibrillar proteins (except MLC1x) on a silver-stained gel after SDS-PAGE. Skinned (S) and non-skinned (NS) segments of the same fibres (fibre type indicated below each lane) are shown in adjacent lanes for visualization of the bands containing soluble proteins (i.e. the bands present in non-skinned segments but absent from skinned segments). For clarity, a type 1 fibre only was used to represent the fast twitch fibres, although significant differences in the quantitative and qualitative expression of MLC and troponin isoforms did exist among the twitch fibres. Note that skinning removed a prominent soluble protein band just above the MLC1 region in both fibre types. Skinning also removed a prominent soluble protein band between MLC1f and TnCf, as well as a band below TnCT in tonic fibres. Note that this frog did not contain the MLC1x isoform in any of its fibre types.
|
|
View larger version (20K):
[in a new window]
|
Fig. 5. The MLC3/MLC1 ratio increased in proportion to the percentage of type 1 MHC in single fibres, but was highly variable within fibre types. (A) MLC3/MLC1 ratio and MHC isoforms were measured in single type 1, type 1-2 and type 2 fibres by quantitative densitometry of gels after SDS-PAGE. Fibres (N=100) were obtained from the anterior tibialis (AT), cruralis (CR), gluteal (GL), gracilis (GR), semimembranosus (SM) and sartorius (SA) muscles. Each symbol corresponds to a different muscle. Linear regression showed that the MLC3/MLC1 ratio increased in direct proportion to the percentage of type 1 MHC. There was substantial variability in MLC3/MLC1 ratio within each fibre type, and the highest values were all found in the GR muscle. (B) MLC3/MLC1 ratio for fibres that contained MLC1x (N=16; data are subset of A). The relationship between the MLC3/MLC1 ratio and percentage of type 1 MHC was similar to A, and the full range of fibre types was represented.
|
|
View larger version (10K):
[in a new window]
|
Fig. 6. MLC1x/MLC1f was higher in type 2 than type 1 fibres. The molar ratio of MLC1x/MLC1f was measured in single type 1, type 1-2 and type 2 fibres by quantitative densitometry of gels after SDS-PAGE. Linear regression showed that MLC1x/MLC1f ratio decreased in direct proportion to the percentage of type 1 MHC (slope=–0.003; r2=0.288; P=0.03).
|
|
View larger version (34K):
[in a new window]
|
Fig. 7. MHC and MLC isoform composition varied substantially along the length of single fibres. MHC isoform content and MLC3/MLC1 ratio were determined in 1 mm segments along the full length of single fibres (N=10) by quantitative densitometry of gels after SDS-PAGE. All fibres were obtained from the anterior tibialis muscle. Each letter (A–J) represents a different fibre.
|
|
View larger version (12K):
[in a new window]
|
Fig. 8. The MLC3/MLC1 ratio increased in proportion to the percentage of type 1 MHC in individual 1 mm segments along the length of fibres. MLC3/MLC1 ratio plotted versus the percentage of type 1 MHC for all segments from each of the 10 single fibres shown in Fig. 7. Linear regression analysis showed that the MLC3/MLC1 ratio increased in direct proportion to the percentage of type 1 MHC.
|
|
© The Company of Biologists Ltd 2001
