Cilia, Centrosomes and Skeletal Muscle

Abstract

Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.

Keywords: cytoskeleton; differentiation; extracellular matrix; myogenesis; primary cilia; proliferation; satellite cells.

Figure 1

Primary cilia assembly and disassembly during myogenesis. (A) The mother centriole with distal appendages forms the basal body of primary cilia. The distal appendages delineate a transition zone and a ciliary compartment distinct from the cytoplasm. Axonemes are comprised of bundles of microtubules that extend upwards from the basal body and are encased in a ciliary membrane. (B) Cross-sectional view of axoneme. Motile cilia possess a 9 + 2 radial configuration of axonemal microtubules. Outer doublet microtubules are connected by nexin links and interact with central single microtubules via radial spoke complexes. Non-motile primary cilia in muscle are organised in a 9 + 0 configuration. (C) Cilia formation and resorption during myogenesis. Satellite cells expressing PAX7 (PAX7^+ve) are ciliated, and these are resorbed as satellite cells specify proliferative myoblasts that express Myf5. Cilia are briefly reassembled in myoblasts as they downregulate PAX7, upregulate MYOD (MYOD^+ve) for differentiation and exit the cell cycle. During late stage differentiation and fusion into myotubes, cilia are disassembled, and centrosomes undergo reduction. Cilia are largely absent in mature myofibers but found on quiescent satellite cells located between the myofiber and basal lamina.

Figure 2

Centrosome reduction during myoblast differentiation. The inactivation of and reduction in centrosomes, which serve as basal bodies for cilia, accompany the terminal differentiation of myoblasts and fusion into myotubes. Centrosome volumes are reduced and pericentriolar material, comprising microtubule anchoring/nucleating complexes, are redistributed to other cellular compartments. In addition, proteins that tether centriole pairs are severed, and centriole barrels are disassembled.

Figure 3

Hedgehog signalling on primary cilia. (A) In the absence of Sonic hedgehog (SHH), Patched1 (PTCH1) inhibits Smoothened (SMO) and prevents SMO localisation to the primary cilium. Suppressor of Fused (SUFU) prevents activation of full-length GLI (GLI-FL) transcription factor, leading to proteosome processing of GLI-FL to its repressor form (GLI-R). GLI-R translocates into the nucleus to repress the expression of SHH target genes. (B) In the presence of SHH, PTCH1 releases its inhibition to SMO. SMO is then activated and localises to the primary cilium along with KIF7, SUFU and full-length GLI (GLI-FL). Activated SMO allows SUFU to release GLI-FL at the tip of the cilium to allow activation of GLI (GLI-A). GLI-A then translocates into the nucleus and transcribes SHH target genes, such as GLI1, PTCH1 and Myogenic differentiation 1 (MYOD).

Figure 4

Diagram of skeletal muscle indicating cilia of a satellite cell on a myofiber surface and cilia of various cells in interstitial connective tissue. In the adult skeletal muscle, the quiescent satellite cell is on the myofiber and under the basal lamina. The myofiber is surrounded by the extracellular matrix, which also contains blood vessels, nerves, macrophages, fibroblast/fibro–adipogenic progenitor (FAP), adipocytes and myofibroblasts.

Figure 5

Cilia resorption and cilia remnants in newly formed myotubes. Tissue cultures of C2C12 mouse skeletal myoblasts during (A) proliferation (cultured in 10% fetal bovine serum) and (B) after 3 days in differentiation medium (2% horse serum) to induce fusion to form myofibers. Cells are immunostained to show cilia (green Arl13b) and basal body (red γ-tubulin) in (i) and (ii), respectively, or in (iii) only for cilia (green Arl13b) and counterstained with actinin (red) to show differentiating myoblasts and myotube sarcoplasm. Cells observed by fluorescent microscopy. Primary cilia are evident in (A) undifferentiated (actinin^−ve), mono-nucleated myoblasts. Cilia are disassembled in (B) as myoblasts differentiate (actinin^+ve) and fuse into multinucleated myotubes. Line arrows indicate cilia in myoblasts. Arrowheads indicate cilia remnants in myotubes. Scale bars = 20 μm.