Unique matrix structure in the rough endoplasmic reticulum cisternae of pseudoachondroplasia chondrocytes

Abstract

Mutations in cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Because COMP exists as a homopentamer, only one mutant COMP subunit may result in an abnormal complex that is accumulated in expanded rough endoplasmic reticulum (rER) cisternae, a hallmark of PSACH. Type IX collagen and matrilin-3 (MATN3), also accumulate in the rER cisternae of PSACH chondrocytes, but it is unknown how mutant COMP interacts with these proteins. The studies herein focus on defining the organization of these intracellularly retained proteins using fluorescence deconvolution microscopy. A unique matrix organization was identified in which type II procollagen formed a central core surrounded by a protein network of mutant COMP, type IX collagen, and MATN3. This pattern of matrix organization was found in multiple cisternae from single chondrocytes and in chondrocytes with different COMP mutations, indicating a common pattern of interaction. This suggests that stalling of mutant COMP and an interaction between mutant COMP and type II procollagen are initiating events in the assembly of matrix in the rER, possibly explaining why the material is not readily cleared from the rER. Altogether, these data suggest that mutant COMP initiates and perhaps catalyzes premature intracellular matrix assembly.

Figure 1

ECM proteins COMP, types II and IX collagen, and MATN3 in the human growth plate and cultured normal and PSACH, G427E, D469del, and D511Y cartilage nodules. Chondrocyte nodules were treated as described previously and in Materials and Methods. Proteins are shown: type II collagen (yellow), COMP (green), type IX collagen (red), and MATN3 (blue). Types II and IX collagens, COMP, and MATN3 co-localize in the matrix, indicating similar distributions in the NGP (A–E) and normal cartilage nodules (F–J). Type II collagen is minimally diminished in the PSACH matrices (K, P, U). The three PSACH nodules demonstrate markedly diminished COMP, type IX collagen, and MATN3 staining (L–O, Q–T, V–Y). Intracellular retention of all four proteins can be appreciated and are shown as dots in all of the PSACH chondrocytes. Scale bar = 20 μm.

Figure 2

ECM and expanded rER cisternae in PSACH chondrocytes compared with normal chondrocytes. Chondrocytes and surrounding matrix images were captured and modeled as described in Materials and Methods and presented as wire-frame renditions. Proteins shown: type II collagen (yellow), type IX collagen (red), COMP (green), and MATN3 (blue). One representative chondrocyte is shown in each panel with the nuclei removed from the picture. The NGP (A) and cultured nodule chondrocytes (B) show a layering and interweaving of all four ECM proteins in the matrix only. In contrast, the PSACH chondrocytes (C–E), show abundant type II collagen in the matrix relative to the other three proteins and to normal controls. All of the PSACH chondrocytes have enlarged rER cisternae containing all four proteins, which are depicted by wire frame. Scale bar = 5 μm.

Figure 3

Arrangement of ECM proteins in the rER of PSACH chondrocytes. Images of rER were captured using fluorescence deconvolution microscopy and modeled as described in Materials and Methods and shown here as wire-frame renditions. Proteins shown: type II collagen (yellow), type IX collagen (red), COMP (green), and MATN3 (blue). rER cisternae from chondrocytes with G427E (A–E), D511Y (F–J), and D469del (K–O) mutations. The retained proteins in the rER have a similar intracellular structure composed of a central core of type II procollagen (A, E, F, J, K, and O) that is surrounded by COMP, type IX collagen, and MATN3 (B–E, G–J, L–O). This pattern is observed with each of the COMP mutations. Scale bar = 2.5 μm.

Figure 4

Combined wire-frame and solid models of ECM proteins in PSACH rER. The rER cisternae images in Figure 3, J and O, with D469 del and D511Y mutations, respectively, were rotated to visualize COMP, types II and IX collagen, and MATN3. Proteins are shown: type II collagen (yellow), type IX collagen (red), COMP (green), and MATN3 (blue). Cisternae from nodules with D511Y (A–C) and D469del (D–F) COMP mutations are shown. Wire-frame renditions are in A and D. Type II collagen is seen as a solid central core in both cisternae (B, C, E, and F). Two representative cisternae, demonstrating the matrix network surrounding the type II collagen core, are shown. Two small cisternae are developing, and the network is assembling around the type II procollagen core (B and C). Scale bar = 2.5 μm.