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Myofibrillar protein isoform expression is correlated with synaptic efficacy in slow fibres of the claw and leg opener muscles of crayfish and lobster

Donald L. Mykles^1,*, Scott Medler¹, Annette Koenders² and Robin Cooper³

¹ Department of Biology, Cell and Molecular Biology Program, Program in Molecular, Cellular and Integrative Neurosciences, Colorado State University, Fort Collins, CO 80523, USA,
² School of Natural Sciences, Edith Cowan University, Joondalup Drive, Joondalup, WA 6027, Australia and
³ Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington, KY 40506, USA

View larger version (44K): [in a new window]   Fig. 1. Opener muscle in the crayfish walking leg showing regional variation in excitatory postsynaptic potential (EPSP) amplitudes. The excitatory motor neuron was stimulated at 30 Hz (10 stimuli per train). The largest potentials were measured in proximal fibres, with smaller potentials in distal fibres; the smallest potentials occurred in central fibres. The double-headed arrows indicate where the medial EPSP traces originated on the muscle. The numbered arrows indicate where the lateral EPSP traces were sampled and correspond to the numbered traces on the right. The regional differences are similar for both lateral and medial halves of the muscle.

View larger version (46K): [in a new window]   Fig. 2. Opener muscle in the crayfish claw showing regional variation in excitatory postsynaptic potential (EPSP) amplitudes. The excitatory motor neuron was stimulated at 50 Hz (20 stimuli per train). The largest potentials were measured in proximal fibres, with smaller potentials in distal fibres; the smallest potentials occurred in central fibres. The double-headed arrows indicate where the medial EPSP traces originated on the muscle. The numbered arrows indicate where the lateral EPSP traces were sampled and correspond to the numbered traces on the right. The regional differences are similar for both lateral and medial halves of the muscle.

View larger version (11K): [in a new window]   Fig. 3. Facilitation indices (FIs) for crayfish claw opener muscle. Trains of stimuli at 50 Hz were given to produce 20 events, which were used to calculate FIs from the ratios of the amplitudes of the fifth, tenth, fifteenth and twentieth excitatory postsynaptic potentials. Values of FI are means ± S.E.M.

View larger version (13K): [in a new window]   Fig. 4. Regional variation in excitatory postsynaptic potential amplitudes in the opener muscles in the lobster walking leg (left) and claw (right). The excitatory motor neuron was stimulated at either 30 Hz/10-pulse train (walking leg) or 50 Hz/20-pulse (claw) train. The largest potentials were measured in proximal fibres, with smaller potentials in distal fibres; the smallest potentials occurred in central fibres.

View larger version (33K): [in a new window]   Fig. 5. Analysis of the myofibrillar protein isoform composition of lobster claw opener muscle by SDS–PAGE and western blotting. Fast (F), slow-twitch (S1) and slow-tonic (S2) fibres from lobster cutter claw closer, crusher claw closer and superficial abdominal muscles are provided for reference (lanes a–c, respectively). Claw opener fibres are arranged according to their position from most proximal (lane d) to most distal (lane i). (A) Silver-stained polyacrylamide gel showing myofibrillar proteins (MHC, myosin heavy chain; P, paramyosin; P75, 75 kDa protein; TnT, troponin-T; Ac, actin; Tm, tropomyosin; TnI, troponin-I). (B) Western blot probed with anti-TnT antibody. S2 fibres (lanes c–e, i) possess varying amounts of TnT1 (arrow), with higher levels in proximal fibres. (C) Western blot probed with anti-P75 antibody. Only fast fibres possess the P75 protein (lane a). (D) Western blot probed with anti-TnI antibody. Although not all five isoforms were detected at this loading, there were clear differences between F (lane a), S1 (lanes b, f–h) and S2 (lanes c–e, i) fibres. On the basis of this analysis, the proximal (lanes d, e) and distal (lane i) fibres were classified as S2, while central fibres (lanes f, g) were classified as S1. The positions of molecular mass standards are indicated on the left.

View larger version (22K): [in a new window]   Fig. 6. Western blots of crayfish and lobster opener muscle fibres probed with an antibody to troponin-T (TnT) . Fast (F), slow-twitch (S1) and slow-tonic (S2) fibres from lobster cutter claw closer, crusher claw closer and superficial abdominal muscles are provided for reference (lanes a–c, respectively); the reference fibres used for the lobster blot (A) were different from the reference fibres used for the crayfish blots (B,C). In lobster, only the proximal fibres (S2) in the leg opener muscle expressed TnT1 (A, lanes d, e). The proximal and distal fibres (S2) expressed variable amounts of TnT1 (arrow) in the crayfish leg opener (B, lanes d, e, h, i) and crayfish claw opener muscles (B, lanes d, e, g), with higher levels of TnT1 in proximal fibres. The central region of crayfish opener muscles (B, lanes f, g; C, lane f) and the central and distal regions of lobster leg opener (A, lanes f–h) contained S1 fibres. The positions of molecular mass standards are indicated on the left.