Teething during sleep: Ultrastructural analysis of pharyngeal muscle and cuticular grinder during the molt in Caenorhabditis elegans

Abstract

Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.

Fig 1. C. elegans pharyngeal grinder is morphologically changed during the L4-to-adult molt.

The DIC Images at different times after pumping cessation. Anterior is to the right. The terminal bulb containing the grinder is centered in the field of view. Filled white arrows point to the L4 grinder and unfilled arrows point to the nascent adult grinder. Scale bar = 5 μm.

Fig 2. Transmission electron micrographs of the medial terminal bulb region in animals at various indicated times post pumping cessation (PC) or at the pre-lethargus L4.7 stage.

The larval pharyngeal cuticle is separated from pharyngeal muscle cells and degraded through the first hour of the molt. At the same time, pharyngeal muscle cells adopt an epithelial secretory character, distinct from their contractile cell character, to synthesize and assemble an adult grinder. Anterior to the right. Scale bars = 1 μm.

Fig 3. The grinder is composed of five layers of extracellular components.

(A) Transmission electron micrograph of the grinder in an active 1^st day adult animal. (B) Pseudocoloring of image shown in (A). (C) Cartoon line drawing generated from psuedocoloring shown in (B). The names of the five layers and of the intracellular and luminal spaces are indicated in the color key. Scale bar = 200 nm.

Fig 4. Pharyngeal myoepithelial cells pm6 and pm7 transdifferentiate during lethargus into secretory cells.

TEM Image of the grinder at the active L4.7 stage (A), 10 min post-PC stage (B), and 2.5 hr post-PC stage (C). For each stage, the top row (i) shows the TEM, the middle row (ii) shows pseudocoloring of the TEM, and the bottom row (iii) shows an area of pm6 subjacent to the grinder. In the pseudocolored images (ii), light brown denotes the terminal bulb lumen, yellow denotes the L4 grinder, cyan denotes the separation space between the larval grinder and pharyngeal myoepithelial cells, purple denotes pm6 papillae, pink denotes pm6, and navy denotes pm7. Scale bar = 1 μm in the top 2 images in all three panels A-C. (Aiii) L4.7 animals have uninterrupted muscle striations (highlighted by white dash boundaries) connected to the plasma membrane below the apical membranes of pm6 and pm7. Scale bar = 100 nm in Aiii. (Bii and Biii) In the 10-min post-PC terminal bulb, the area between the apical membranes of of pm6 and pm7 and ~500 nm below the apical membranes is the interruption zone or IZ (bounded by white dashed lines. High magnification view of the area outlined in Bii shows interruption of muscle striations; dense core vesicles predominate in this area of pm6. Scale bar = 100 nm for Biiib (b) High magnification view of the box shown in b ii, showing muscle striations (in between parallel white dashed lines) in an area located posterior of the IZ in pm6. Scale bars = 100 nm. (Ciii) High magnification view of the box in (Cii), showing restoration of cell-to-cuticle and muscle striation attachments to the basal-side of the adult grinder (muscle striations are bounded by white dashed lines). Scale bar = 200 nm in Ciii.

Fig 5. Vesicular morphology changes during lethargus, coincident with L4 grinder dissolution and adult grinder construction.

(A) Transmission electron micrograph of an animal 30 minutes post-pumping cessation (PC). Insert: High magnification view of vesicular constituents subjacent to the grinder in pm6, as well as part of pm7. The top-half of the image shows the shed L4 grinder as well as the developing adult grinder, and the bottom-half shows pm6 and pm7 cells. The pm6 and pm7 cells are separated by plasma membranes. A dense core vesicle, denoted by a black arrowhead, appears set to fuse with the plasma membrane. A clear core vesicle is denoted by a white arrowhead, and a crescent-rimmed clear core vesicle is denoted by a black-outlined white arrowhead. Scale bars = 1 μm for the left image and 200 nm for the right image. (B) Cartoon representations of the vesicular types observed in terminal bulb muscle cells during lethargus.

Fig 6. L4 grinder degradation begins rapidly after feeding cessation, coincident with the appearance of abundant dense core vesicles.

(A) Active L4.7 and (B) five min post-PC. (i) TEM of the terminal bulb lumen, L4 pharyngeal grinder, pharynx muscle cell 6 (pm6,) and part of pharynx muscle cell 7 (pm7). (ii) Pseudocoloring, light brown denotes the terminal bulb lumen, yellow denotes the L4 pharyngeal grinder, pink denotes pm6, purple denotes pm6 papillae, red denotes the bottom boundary of L4 pharyngeal grinder, and navy denotes pm7. (iii) Cartoon using the color scheme used in (ii) with the addition of orange denoting plasma membranes. In the L4.7 terminal bulb, vesicular activity is evident in both pm6 and pm7, though there are more vesicles in the 5 min post-PC pm6 and pm7 cells. Anterior is up. Scale bar = 1 μm.

Fig 7. The adult grinder is formed through the sequential addition of layers, coincident to displacement and degradation of the L4 grinder.

(A) 10 minutes post-PC and (B) 15 min post-PC. (i) TEM of the terminal bulb. (ii) Pseudocoloring. Light brown denotes the terminal bulb lumen, yellow denotes the L4 pharyngeal grinder, red denotes the bottom boundary of L4 pharyngeal grinder, cyan denotes the separation space, blue denotes the upper boundary of pm6, purple denotes pm6 papillae, pink denotes pm6, and navy denoting pm7. (iii) Cartoon using the color scheme used in (ii) with the addition of plasma membranes shown in orange. Anterior is up. Scale bar = 1 μm.

Fig 8. Layers 3–5 of the L4 grinder are fully dissolved as the adult grinder forms.

(A) 30 minutes post-PC and (B) 60 minutes post-PC. (i) TEMs of the terminal bulb (ii) Pseudocoloring. Light brown denotes the terminal bulb lumen, yellow denotes the solubilizing L4 pharyngeal grinder, red denotes the lining the new adult luminal layer, blue denotes layer 2, green denotes layer 3, gold denotes layer 4, purple denotes pm6 papillae, pink denotes pm6, and navy denotes pm7. (iii) Cartoon representation using the color scheme used in ii, with the addition of plasma membranes denoted by orange. Anterior is up. Scale bar = 1 μm.

Fig 9. The formation and maturation of the adult grinder.

(A) 2.5 hours post-PC (B) 1st day adult. (A and B) 2.5 hours post-PC and 1st day adult, respectively. (i) Transmission electron micrograph of the terminal bulb. (ii) Pseudocoloring. Light brown denotes the terminal bulb lumen, red denotes the lining the new adult luminal layer, blue denotes layer 2, green denotes layer 3, gold denotes layer 4, neon green denotes the pericellular layer, purple denotes pm6 papillae, pink denotes pm6, and navy denotes pm7. (iii) Cartoon representation using the same color scheme used in ii, with the addition of orange, which denotes plasma membranes. Vesicles are not shown, but sparse appearances of clear core are evident. Anterior is up. Scale bar = 1 μm.

Fig 10. The astacin metalloprotease NAS-6 is required for digestion of the larval cuticle.

Light green denotes hypodermal cells, pink denotes the terminal bulb, red denotes the adult grinder and pharyngeal cuticle lining, cyan denotes the terminal bulb lumen space where several E. coli bacteria are trapped, yellow denotes the ecdysed L4 cuticle, forest green denotes the pharyngeal-intestinal valve cells, and blue denotes the intestine. Regions not colored indicate the pseudocoelomic cavity. This micrograph of a nas-6 mutant shows that the L4 cuticle was not digested and the alimentary passage was occluded by the undigested L4 cuticle, trapping intact bacteria in the terminal bulb and impairing mechanical disruption of bacteria. Anterior is up. Scale bar = 1 μm.

Fig 11. The aged grinder is worn down and permissive of transport of intact bacteria to the intestine.

Pseudocoloring of a day-8 adult animal. Pink denotes the pharyngeal cells of the terminal bulb, red denotes the worn, adult grinder and pharyngeal cuticle, and cyan denotes the terminal bulb lumen and part of the intestinal space entrance (left,) where several intact E. coli bacteria are seen in close vicinity to the grinder teeth. Anterior is to the right. Scale bar = 2 μm.