Biochimica et Biophysica Acta (BBA) - General Subjects
Interactions of enolase isoforms with tubulin and microtubules during myogenesis
Introduction
Enolase is a dimeric glycolytic enzyme that catalyses the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP). In higher vertebrates this enzyme exhibits cell-type specific isoforms [1], [2]. During ontogenesis, transitions occur in cell types with high-energy requirements, from αα to ββ in striated muscle cells, from αα to γγ in neurons [3], [4], [5]. The unique embryonic form, αα enolase, remains expressed in most adult tissues and is the ubiquitous isoform. The three isoforms (αα, ββ and γγ) exhibit very similar catalytic properties [6], [7]. Enolase sequences have been well conserved through evolution and sequence identity between the mammalian isoenzymes is ∼ 82% [8]. Three different genes, ENO1, ENO3 and ENO2, encode the α, β and γ enolase subunits, respectively [9], [10]. Specific sequences in promoters of these genes are responsible for the cell type-specific expression of enolases. The ENO1 gene encodes not only the α enolase subunit but also a functionally unrelated protein with a sequence corresponding to the C-terminal region of the α enolase subunit. This protein acts as a transcriptional repressor of the Myc proto-oncogene and has been called myc binding protein 1 (MBP1) [11], [12].
Biochemical and immunocytochemical studies with purified murine enolase isozymes and corresponding specific antibodies indicated the heteroassociations of enolase with other glycolytic enzymes and subcellular particles. The isoforms expressed in striated muscle, the ubiquitous α and the muscle-specific β enolase, interact with glycolytic enzymes of muscle origin [7]. Association of β isoform with troponin but not with actin was detected by in vitro assay [7]. Binding of α and β enolases to the sarcomeric contractile apparatus was demonstrated as well [13].
Expression of the different enolase isoforms during myogenesis has been studied in our and other laboratories [2], [3], [4], [14]. The specific expression of β subunit was detected in the heart of 3-week-old human embryos and in the myotomal compartment of somites from 4-week-old embryos [4], [15]. During ontogenesis, striated muscle differentiation is accompanied by an increase in β enolase expression and by a decrease in the expression of the α isoform [14], [15]. Studies with in vitro cell cultures revealed that β enolase was expressed at low levels in proliferating myoblasts from both embryonic and post-natal muscles, but not at all in cells of non-myogenic lineage. Myoblast fusion is accompanied by a large increase in β enolase expression [15], [16] and concomitant reorganization of the microtubular network (see [17]).
In this work we demonstrated the intracellular expression and localization of enolase isoforms during the differentiation of myoblasts issued from adult muscle stem cells, the so-called satellite cells, into myotubes. We quantitatively characterized the association of enolase isoforms to tubulin using purified proteins and muscle extract, and monitored the co-localization of enolase isoforms with the microtubule filaments.
Section snippets
Enolase and tubulin preparations
Enolase isoforms were purified from mouse brain (αα) and mouse striated muscles (ββ) as described [6], [7] and stored at − 80 °C at 1–4 mg/ml concentration. Tubulin, depleted of microtubule associated proteins (MAP), also called MAP-free tubulin, was purified from bovine brain as described [18].
Pelleting experiments
Tubulin was dialyzed in 50 mM 2-[N-morpholino]ethanesulfonic acid buffer (pH 6.8) at 4 °C for at least 3 h, then centrifuged at 4 °C at 100 000×g for 20 min. The supernatant was polymerized into
Distinct localization of enolase isoforms in myoblasts and myotubes
Primary cell cultures have been derived from the quiescent satellite cells present on rat hind limb [19] and used to investigate the sub-cellular localization of enolase isoforms by immunofluorescence confocal microscopy. High specificity of antibodies directed against each enolase isoform had previously been ascertained [6], [7]. In the 5-day cultures both myoblasts (undifferentiated muscle cells) and myotubes (differentiated cells, about 5% of the total cell population) were present. Fig. 1
Role of tubulin organization in the subcellular localization of enolase isoforms
Formation of myotubes by fusion of myoblasts is accompanied by a large increase in β enolase expression [15], [16]. Indeed, Western blotting analyses indicated that the β enolase level was increased significantly, while the α enolase level did not change after 10 days differentiation of satellite cells. Our present data, as summarized in Table 1, reveal that the subcellular localization of enolase isoforms is connected with the organization of the microtubule system during in vitro myogenesis.
Acknowledgements
This work was supported by grants from CNRS-ASH #11971 and from Balaton #08972SG, by the Hungarian National Scientific Research Fund Grants OTKA T-046071 and T-049247, by the Hungarian Ministry of Education Grant OMFB-00346/2005, FP6–2003-LIFESCIHEALTH-I: Bio-Sim, NKFP-MediChem2 1/A/005/2004.
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