Ultrastructural Modelling of the Matrix-Cilium-Golgi Continuum in Hyaline Chondrocytes
Overlooked for decades as an evolutionary vestigial organelle, the primary cilium has recently become the focus of intensive investigations into understanding of the physical structure and processes of eukaryote cell function. The cilium is central to various signalling pathways and modalities for signalling, allowing centrosomal processing and regulation of cellular and organelle function. The enigmatic processes of their nanoscale ciliogenesis and sensory function at present remain poorly understood. Dysfunction of their normal function or component proteins correlates with a wide spectrum of diseases, or ciliopathies, which express as developmental disorders and pathologies . Normal connective tissue function requires cells to sense and respond to mechanical and physiochemical changes in the extracellular environment, and matrix, in order to maintain their form and function. In hyaline cartilage, chondrocyte primary cilia are located at an intermediate position between the mechanically functional extracellular matrix (ECM) surrounding the cell, and the intracellular organelles responsible for producing, modifying, transporting and secreting extracellular matrix materials [26, 96]. While many cellular and physiological processes involving primary cilia are at present known, accurate in situ knowledge of their form and fine structure in connective tissue is lacking. A three dimensional model has been created of an in situ chondrocyte primary cilium. This details the anatomical structure of the cilium and its relationship to other cellular organelles. Components were mapped and divided into groups containing: the Matrix, the Cilium, the Centrosome, the Golgi apparatus and the Nucleus. The extracellular matrix was found to consist of interconnected tethered proteoglycans and matrix granules, with the granules composed of aggregates of finer components. These are tethered to the ciliary membrane at localised binding points. The membrane itself was observed as a dynamic extension of the cell membrane, fitting neatly over the axoneme microtubule doublets and their transport cargoes, while responding to the bending and torsional forces induced by the ECM. The ciliary axoneme comprises a ‘9+0 structure’ of microtubule doublets of various lengths and inclinations, associated linkage proteins, ciliary necklace proteins, and intraflagellar-transport particle ‘rafts’ of materials, contained within the ciliary membrane. The basal body is composed of nine microtubule triplets, and is decorated with appendages of basal feet, for the attachment of radiating cytoplasmic microtubules (as the foci of the microtubule organising centre, MTOC), as well as alar sheets and transition fibres, which function to anchor the basal body to the cell and ciliary membrane. The basal body microtubule polarisation, curl and inclination have been determined, along with those of the subtending proximal centriole, together the constituent components of the centrosome. The nuclear double membrane, and nuclear pores, and their close co-relation with the centrosome are shown. Golgi cis-, medial- and trans-compartments, with associated transport vesicles are described along with their polarization. Clathrin coated pits were found, indicating receptor-mediated endocytosis. The outcome of this study is the first high-resolution reconstruction of a primary cilium. The model will enable interpretation of the interactions and involvement of the many biochemical and biophysical pathways now known to be associated with the cilium, and will lead to new understandings of processes fundamental to the workings of the eukaryotic cell. As a schematic, the model raises new questions about the visualisation of the structure and form of the primary cilium. A vast volume of literature exists upon many aspects of cell biology, however few studies have undertaken investigations of primary cilia to understand their structure, and attempt to translate it to function. This is the first study to attempt to probe the complex relationship between the extracellular matrix, the mechanosensitive primary cilium, the centrosome and the Golgi apparatus, which as a continuum are responsible for maintaining the cells microenvironment.
Advisor: Walker, Robert
Degree Name: Doctor of Philosophy
Degree Discipline: Medicine
Publisher: University of Otago
Keywords: Primary; Cilium; Matrix; Golgi; Electron; Tomography; Modelling
Research Type: Thesis