High‐resolution imaging of muscle attachment structures in Caenorhabditis elegans

Abstract

We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in C. elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane (UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin) and UNC-95), are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal “zipper-like” structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in atn-1 mutants that are null for α-actinin and lack the deeper extensions of dense bodies, showed “zipper-like” structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the “zipper-like” structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have “dot-like” dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape.

The Rho-GEF PIX-1 directs assembly or stability of lateral attachment structures between muscle cells

“…2 (top row), there is clearly a thin band of F-actin that lies near the muscle cell boundary (indicated by yellow arrow), which can be identified by three criteria: (1) its location between two adjacent spindle-shaped body wall muscle cells (Supplementary Fig. 2 ), (2) being thinner than a typical I-band which alternates with myosin A-bands, and (3) not projecting throughout the depth of the myofilament lattice 5 like a typical I-band, which can be discerned by observing less intense signal when the optical slice is taken deeper into the lattice (the label of deeper part in Fig. 2 and Supplementary Fig.…”

“…Closer examination of confocal images of pix-1(gk893650) shows that the boundary defect is more subtle in this mutant than in the nonsense and intragenic deletion pix-1 mutants. In Qadota et al 5 , we reported that by structured illumination microscopy (SIM), in wild-type muscle, the boundaries appear like a zipper, in which the two sides of the zipper are closely apposed to each other (as if the zipper were closed). Zoomed-in confocal views of muscle ( ; sfIs20] in which the muscle cell boundary defect observed in the nonsense mutant pix-1(gk299374) has been rescued by an integrated array of wild-type pix-1 expressed from a muscle-specific promoter (see Fig.…”

“…This architecture, together with what is known about the molecular composition of these structures, indicates that nematode dense bodies and M-lines also act as costameres. C. elegans body wall muscle also has IACs at the muscle cell boundaries 5 , where they form attachment plaques that anchor the muscle cell to a thin layer of ECM that lies between adjacent muscle cells.…”

Investigating the correlation of muscle function tests and sarcomere organization in C. elegans

“…Unlike vertebrates, the contractile forces in C. elegans are mostly transmitted laterally to the cuticle resulting in bending and sinuous locomotion of the worm on an agar surface (14). In addition, integrin adhesion complexes exist at attachment plaques lying at the boundaries between adjacent muscle cells separated by basement membrane (15), and these also are likely to be involved in force transmission as their absence results in a locomotion defect (16).…”

“…The conserved molecular pathways contributing to sarcomere structure, and muscle function have been studied in C. elegans using a variety of methods. The animal's transparent body along with immunostaining approaches and various microscopy techniques has helped to visualize the muscle structure (15,(17)(18)(19)(20)(21). Impairments in muscle function due to genetic defects have been studied by observation of gross phenotypes such as "Unc" (Uncoordinated) adults and "Pat" (Paralyzed arrest at 2-fold) embryonic lethals (13,17,(22)(23)(24).…”