Interactions of enolase isoforms with tubulin and microtubules during myogenesis

Abstract

Enolase is a glycolytic enzyme, expressed as cell-type specific isoforms in higher vertebrates. Herein we demonstrated for the first time that enolase isoforms interact with microtubules during muscle satellite cell differentiation. While in undifferentiated myoblasts the ubiquitous αα enolase isoform, expressed at high level, exhibited extensive co-localization with microtubules, the muscle-specific ββ isoform, expressed at low level, did not. During differentiation, the level of β subunit increased significantly; the α and β enolase immunoreactivities were detected both in cytosol and along the microtubules. We identified tubulin from muscle extract as an interacting protein for immobilized ββ enolase. ELISA and surface plasmon resonance measurements demonstrated the direct binding of enolase isoforms to tubulin with an apparent K_D below the micromolar range, and indicated that the presence of 0.8 mM 2-phosphoglycerate abolished the interaction. Our data showed that, at various stages of myogenic differentiation, microtubules were decorated by different enolase isoforms, which was controled by the abundance of both partners. We suggest that the binding of enolase to microtubules could contribute to the regulation of the dynamism of the cytoskeletal filaments known to occur during the transition from myoblast to myotubes.

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.