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The decrease of the cytoskeleton tubulin follows the decrease of the associating molecular chaperone B-crystallin in unloaded soleus muscle atrophy without stretch

Department of Life Sciences, The Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan

¹ Correspondence: Department of Life Sciences, The Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan. E-mail: atomi{at}idaten.c.u-tokyo.ac.jp

SPECIFIC AIMS

The cytoskeletal component tubulin/microtubule (MT) commonly allows the cell to respond mechanically to the environment, and the concentration of free tubulin dimer is autoregulated in the balance of free dimer and polymeric forms of MT protein, having an intrinsic property of "dynamic instability." To explore causal relationships between the molecular chaperone B-crystallin and cytoskeletal structure, we examined tubulin changes that were related to B-crystallin, including associations between the two proteins, using immunoprecipitation and taxol-dependent MT precipitation, in rat soleus muscle extracts, and mouse C2C12 cultured muscle cells.

PRINCIPAL FINDINGS

1. Tubulin protein content decreased with B-crystallin during hindlimb suspension (HS), and its decrease was prevented by passive stretch of the hindlimb; a similar effect was seen for B-crystallin
The content of MT is higher in slow-twitch skeletal muscle than in fast-twitch skeletal muscle as well as B-crystallin, which decreases early and dramatically during muscle atrophy. B-Crystallin is considered to play a role in the maintenance of cytoskeletal components, in both the free and filamentous forms. It colocalizes with intermediate filaments and MTs in myoblast cells. We measured the content of tubulin in rat soleus muscle, which consists of mostly slow-twitch fibers. A rat HS experiment was carried out to induce muscle atrophy. The atrophied soleus muscle in the HS treatment group (S) exhibited a significant decrease in tubulin content after the treatment and also decreased B-crystallin content, both of which were prevented by the hindlimb passive stretch treatment (ST) (Fig. 1 ). Other heat shock proteins such as heat shock 90 kDa protein (Hsp90), Hsp70, Hsp27, and p20 also decreased with HS, with the exception of heat shock cognate 70 kDa protein (Hsc70).

View larger version (54K): [in this window] [in a new window]   Figure 1. Quantification of tubulin and B-crystallin protein contents in rat soleus muscles during HS, with or without stretch, and changes in levels of other heat shock proteins as measured by Western blot after HS. A) Correlation of relative densitometric units with purified tubulin concentration. B) Tubulin and B-crystallin contents decreased significantly during HS treatment. Levels were restored after 10 days of HS followed by recovery. n = 5. Mean ± SEM. *P <0.05, **P <0.01. C) Immunoblot of other Hsps (Hsp90, Hsc70, Hsp70), sHsps (Hsp27, p20, B-crystallin), and -tubulin during HS. Hsps except Hsc70 decreased during HS.

2. Confirmation of interactions between B-crystallin and tubulin/MT in soleus muscle extracts by immunoprecipitation and MT sedimentation assay
The molecular chaperone B-crystallin occurs in the soluble cytosolic fractions of the soleus muscle extracts. It was precipitated with the cytoskeleton tubulin/MT, as confirmed by an immunoprecipitation method using an anti--tubulin antibody and a taxol-dependent MTs sedimentation assay. The small heat shock proteins (sHsps) Hsp27 and p20 precipitated with B-crystallin and MT, but Hsp90 and Hsp72, which decreased with HS, did not coprecipitate (Fig. 2 ). It seems that the functions of the sHsps that are molecular chaperones, including B-crystallin, differ from other well-known Hsps.

View larger version (45K): [in this window] [in a new window]   Figure 2. Interaction of B-crystallin and tubulin/MTs analyzed by Immunoprecipitation and taxol-dependent MT polymerization. A) The cytosolic fractions of rat soleus muscle extracts were immunoprecipitated with an anti--tubulin antibody and analyzed by immunoblotting with anti--tubulin and anti-B-crystallin antibodies. B) (a) MT fractions were separated by 2D-PAGE. Seven protein spots were positively identified by MALDI-TOF MS analysis. (b) MT fractions were separated by SDS-PAGE. B-Crystallin (arrow) and -tubulin (arrowheads) were detected by immunoblot.

3. The amount of tubulin mRNA did not decrease during HS, whereas B-crystallin mRNA decreased
The presence of B-crystallin and ßI-tubulin mRNA in soleus muscles during HS treatment was confirmed by semiquantitative RT-PCR. Compared with soleus muscle of control rats, B-crystallin mRNA expression in atrophied soleus muscle decreased according to the duration of HS. After 1 day of recovery, B-crystallin mRNA expression increased by 1.8-fold compared with the control. Decreases of B-crystallin mRNA were consistent with the decreases of B-crystallin protein content. On the other hand, ßI-tubulin mRNA expression did not significantly change during HS, whereas 1 day of recovery markedly increased it by 2.1-fold compared with the control. This reveals that the amount of tubulin mRNA remained almost constant, even though the tubulin content continued to decrease in the atrophied soleus muscle.

4. Effect of B-crystallin on myotube formation and MT development during differentiation of mouse C2C12 cultured cell
To investigate a precise role of B-crystallin in vitro, C2C12 myoblast cells transformed with a sense (C2C12SE) or antisense strand (C2C12AS) of B-crystallin cDNA were cultured. C2C12WT and C2C12SE cells fused well whereas C2C12AS cells could not differentiate into myotube at all. The MT networks in C2C12SE cells developed well and the dominant direction of the microtubules was longitudinal. On the other hand, the MT networks in C2C12AS were thin and spread radially rather than longitudinally.

CONCLUSIONS AND SIGNIFICANCE

In eukaryotic cells, especially muscle fibers, spatial and mechanical functions are developed to a very high degree and depend on a remarkable system of filaments known as the cytoskeleton. A peculiar property of the cytoskeleton is that it is dynamically regulated to allow it to develop tension continuously. The cytoskeleton has been well studied in cultured cells but there are few papers dealing with cytoskeletal proteins in the muscle, with the exception of intermediate filament desmin and/or dystrophy. Most sHsp proteins are expressed in striated muscle cells; they are especially abundant in slow skeletal and cardiac muscle cells. The protein B-crystallin is constitutively expressed at high levels in these cells. The role of sHsps as chaperones in skeletal muscle has not yet been shown.

A novel and significant finding reported here is that tubulin and the molecular chaperone B-crystallin interact in slow muscle extracts. Immunoprecipitation and MT-specific cosedimentation were used to demonstrate interactions between these proteins. Results of animal experiments and biochemical analyses using soleus muscle extracts confirmed an essential in vivo role for the B-crystallin in maintaining tubulin dimers and MT networks. Hsp70 and Hsp90 did not participate in these interactions. This is the first demonstration of this important difference in the roles of Hsps in muscle and suggests that some, but not all, may function as molecular chaperones during adaptation processes. The special roles of sHsps provide insight into a potent mechanism controlling skeletal muscle plasticity that may be regulated by changes in protein structures. The key mechanisms regulating induction of muscle atrophy have not been shown, especially those that occur in the early phase of atrophy. Remodeling of the cytoskeleton must be essential for the early phase of the adaptation, which involves orchestrating essential structures in the muscle cell system.

Tubulin/MTs, which have intrinsic dynamic properties, are essential for myotube formation; their functions in skeletal muscle after differentiation are still unknown. In previous cell culture studies, we demonstrated interactions between tubulin/MTs and B-crystallin and the decrease of B-crystallin during early skeletal muscle atrophy, but not under stretched conditions. We show here that, similar to B-crystallin changes, the content of tubulin in soleus muscle was maintained or significantly decreased after HS, with or without passive stretch. Furthermore, B-crystallin was coprecipitated from soleus muscle extracts with an anti--tubulin antibody and with MT assembly using taxol. These data imply an interaction between B-crystallin and tubulin/MTs in muscle tissues. The decrease in B-crystallin content in soleus muscle occurs more rapidly than the reduction in tubulin content, suggesting a role for B-crystallin as a chaperone for cytoskeletal proteins, especially tubulin/MTs.

The amount of B-crystallin mRNA decreased significantly along with the muscle atrophy level, starting from the second day of HS; that of ßI-tubulin mRNA was unchanged throughout HS. It is known that ß-tubulin mRNA expression levels are autoregulated by free tubulin subunits. The unchanged ßI-tubulin mRNA expression level suggests that free tubulin subunit levels are regulated and remain at a constant level even in atrophied soleus muscle, whereas total tubulin content of the cells decreased. Reduced tension of the soleus muscle by HS may cause MT network destruction in shortened soleus muscle tissues.

Cell culture experiments using mouse myoblast C2C12SE and C2C12AS, whose expression of B-crystallin was up- or down-regulated, respectively, showed C2C12SE fused into thick myotubes with dense MT networks; C2C12AS could not differentiate at all. This implies B-crystallin might be essential for the formation of dense and longitudinal MT networks and for myotube formation. These data suggest that B-crystallin is involved in MT network formation during muscle differentiation in C2C12 cells.

Since publication of the classic paper by Toyama et al. showing substitution of actin filaments for MT under taxol treatment after PKC activation in cultured myotubes, few reports have suggested a role for tubulin/MTs in skeletal muscle. We revealed a significant contribution of tubulin/MTs to muscle plasticity. MT and B-crystallin expression is higher in slow muscle than in fast muscle, which undergoes phasic contraction. This endurance type of contractile property in slow muscle may result in a close interactive organization of the cytoskeleton and mitochondria. The correlation among tubulin/MTs, molecular chaperones for the cytoskeleton, and mitochondria is summarized in Fig. 3 . The novel findings presented here provide new clues relating to conjugation of energy metabolism and tensile properties of striated muscles.

View larger version (31K): [in this window] [in a new window]   Figure 3. The molecular chaperone B-crystallin is essential for maintenance of the cytoskeleton tubulin/MAPs-MT in skeletal muscle, which depends on loading/unloading or stretching/shortening of skeletal muscle. The decrease in levels of B-crystallin protein preceded decreases of the tubulin/MTs protein system. The dynamic protein assembly systems of the cytoskeleton appear to interact with the molecular chaperone system that orchestrates the muscle contracting-stretching machinery. These interactions appear to govern protein homeostasis/homeodynamics; our results indicate that slow muscle is dynamically maintained by cytoskeleton-related molecular chaperones, known as sHsps, including B-crystallin.

Although various muscles express many specific proteins that regulate muscle tension and dynamic muscle contraction, the peculiar properties of tubulin/MTs and actin filament systems, which include dynamic instability or treadmilling with polarity, must be essential for muscle adaptation as it involves remodeling of the cellular architecture.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3060fje; doi: 10.1096/fj.04-3060fje

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