Amorphous no more: subdiffraction view of the pericentriolar material architecture

Abstract

The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The pericentriolar material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules and acts as a nexus for molecular signaling. Although great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance on electron microscopy (EM) have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supramolecular organization of organelles in cells.

Keywords: cell cycle; centrosomes; cilia; mitosis; pericentriolar material; super-resolution microscopy.

Figure 1. Subdiffraction fluorescence microscopy view of the PCM architecture

a) Schematic representations of the architectural elements of interphase centrosome. The three layers of organization—centrioles, PCM fibers and matrices—are represented separately to help visualization. The PCM is organized in two major layers of proteins 1) molecular fibers composed of the elongated coiled-coil proteins pericentrin/PLP and Cep152/Asl have their C-termini near the centriole wall and their N-termini extending towards the periphery 2) A PCM matrix composed of Cep215/Cnn, γ-tubulin and Cep192/Spd-2 molecules. See Fig. 2 for model with mitotic PCM. b) Positional mapping of pericentrin domains on interphase human centrosome. Top. Position of antibody/probe positions on human pericentrin with predicted coiled coil regions indicated. Bottom. Measurements of pericentrin diameter derived from 3DSIM micrographs of human cells stained with anti-pericentrin domain specific antibodies. The PACT domain is localized at the C-terminus with a diameter of ~ 200 nm consistent with the measurements electron micrographs. The N-termini is positioned at distance > 500 nm from the center of the centriole. c) 2D projections of averages of aligned 3D volumes of human centrosomes stained with antibodies against the C- and the N-termini regions of pericentrin. Modified from (37)

Figure 2. Nanometer scale organization of the PCM proximal layer

a) STORM images of S2 cell stained with antibodies against PLP, the Drosophila pericentrin orthologue. (Left) End on view. (Right) Side view. Note the quasi ninefold symmetric distribution of PLP and a ~200 nm gap, which coincides with the position of the nascent daughter centriole. b) 2D projections of 3DSIM micrographs of Drosophila S2 cells stained with antibodies against Plk4 and PLP. Note the interleaved distribution of PLP and Plk4 on interphase centrioles. The N-termini of Asl has been shown to have a similar distribution to Plk4 on interphase S2 cells and the two proteins interact directly in vitro. c) Overview of the experimental scheme used for 3D volume alignment and averaging of subdiffraction resolution images. See Box1. Modified from (37)

Figure 3. The PCM architecture during centrosome maturation

Schematic representation of a mammalian centrosome during centrosome maturation. Note the expansion of the PCM proximal layer of interphase cells during G2/M through the formation of an outer matrix of Cep215, pericentrin and Cep192 molecules. γTuRC are embedded within the PCM and promote microtubule nucleation during mitosis. In contrast, Drosophila PCM expansion does not require the pericentrin homologue PLP.