Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

Abstract

Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography–tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.

Introduction

Skeletal muscle is a terminally differentiated tissue consisting of ordered arrays of multinucleated, contractile myofibers. In vertebrates, skeletal muscle is formed during fetal and postnatal development by differentiation of previously specified myoblasts, which irreversibly exit the cell cycle and become committed myocytes. This transition is accompanied by changes in gene expression, growth factor responsiveness and structural protein production (reviewed in [1]). Upon differentiation myocytes subsequently fuse with each other or with existing myotubes to produce contractile syncytial myofibers [2].

In adult vertebrates myogenesis is believed to be primarily carried out by satellite cells, the somatic stem cells responsible for in vivo maintenance and regeneration of skeletal muscle tissue [3], [4]. These cells, which comprise a very small (1–6%) fraction of total muscle-associated nuclei, are defined anatomically by their position between the basement membrane and the sarcolemma of differentiated muscle fibers [3], [5], [6]. In response to injury, otherwise mitotically quiescent satellite cells become activated and proliferate extensively. The resulting population of adult myoblasts will then transit to the site of injury and differentiate into myocytes to replace the damaged myofibers, either by fusion with each other to form new muscle fibers or by fusing into existing post-mitotic muscle fibers [7], [8]. While the satellite cell compartment is repopulated following completion of a cycle of acute regeneration, it remains unclear what the exact cellular source(s) of these new quiescent cells may be: evidence exists for satellite cell self-renewal, either by asymmetric division [9] or stochastic events [10], as well as possible contributions from muscle-associated mesenchymal stem cell populations [9], [11].

The extracellular milieu encountered by newly-activated satellite cells requires that they detect and respond appropriately to a diverse array of rapidly changing stimuli. In addition to the damaged host muscle, local signaling sources would include coincidently damaged connective tissue, vasculature and nervous tissue, as well as infiltrating cells of the immune system [3]. Local extracellular signals would also be expected to vary with time after the initial injury. Thus, critical roles have been demonstrated for many soluble factors and matrix/adhesion molecules in the muscle tissue during satellite cell-mediated muscle repair [12], [13], [14], and there is a significant amount of ongoing investigation into the signaling pathways that function in satellite cells during regeneration.

An area that has not yet been addressed with respect to satellite cell signaling is the possible involvement of higher-order signaling complexes, such as those that have been proposed to assemble in membrane ‘rafts’. Membrane rafts, also known as lipid rafts, are small (10–200 nm), cholesterol and sphingolipid enriched membranes that function to compartmentalize cellular processes [15], [16]. These regions of the plasma membrane play important roles in intracellular protein transport, membrane fusion and transcytosis; they have also been proposed to act as platforms for assembly of signaling complexes, cell-surface antigens and adhesion molecules. Cellular events commonly associated with membrane raft complexes include cell activation, polarization and signaling [17], [18]. In other adult stem cells (i.e., hematopoietic stem cells) membrane rafts are critical for cell cycle regulation and survival [19], [20], however very little is known about signaling pathways mediated by membrane rafts in satellite cells.

In this study, we attempted to isolate and characterize plasma membrane proteins expressed by primary mouse satellite cells, with the goal of prospectively identifying additional signaling pathways that may impinge upon satellite cell activity. Using liquid chromatograpy–tandem mass spectrometry, we identified classes of transmembrane and membrane-associated proteins present in freshly isolated murine satellite cells and enriched in detergent-resistant membrane domains. While surprisingly few of the expected transmembrane receptors were detected above the reliability threshold, multiple proteins associated with adhesion-mediated signaling were identified. Several have not previously been connected with myogenesis, although many have; a significant subset has also been reported to act via membrane raft complexes. One such protein, neural cell adhesion molecule (NCAM), was found to be present and enriched in the detergent-resistant membrane fraction, and was selected for further study.

When examined in heterogeneous populations of adult myoblasts and myocytes, we found NCAM expression to be coincident with the earliest detectable markers of commitment to differentiation. In order to unequivocally differentiate between proliferation-competent myoblasts and committed myocytes, it is common to assay for expression of differentiation-associated proteins such as myogenin, p21, or muscle structural proteins. However, all of these proteins are cytoplasmic or nuclear, and it would be desirable to determine cell status by assaying for a cell-surface epitope, allowing analysis of living unfixed cells. To date, no such marker has been reported. Here we show that by indirect immunohistochemistry, NCAM labels only nonproliferating cells that, based on their expression of differentiation markers including myogenin and muscle creatine kinase have committed to differentiation. We also use NCAM expression to separate a heterogeneous population of adult myoblasts into proliferating and differentiated fractions, as confirmed by expression of either proliferation or pro-myogenesis proteins. This molecular tool therefore represents a novel, non-terminal assay for the early identification and sorting of committed myocytes from heterogeneous populations derived from primary mouse satellite cells.