Microtubules are polymers of αβ-tubulin that have a fundamental role in cell function. In animal cells, the main microtubule organization center is the centrosome, an organelle that is involved in integral tasks within cells of diverse tissues. The centrosome helps establish the axis of cell division, a critical step in stem cell duplication and embryonic development, and is involved in determining the plane of cytokinesis, thus ensuring inheritance of an equal number of chromosomes by daughter cells. In ciliated cells centrosomes dock at the cell membrane and differentiate into basal bodies, which are essential for the formation of the axoneme, a structure critical for integrating signals via primary cilia and for facilitating movement as flagella1–3
. Not surprisingly, mutations associated with several hereditary diseases have been mapped to genes whose products encode centrosomal proteins4
In the current view5–7
the centrosome is composed of two structural elements: the centriole, a barrel-shaped cylinder encircled by microtubule blades and the PCM, described as an amorphous, electron-dense structure surrounding the centrioles. The primary role of the PCM is to anchor microtubules directly or through microtubule nucleating centers (γ-tubulin ring complexes; γTuRC) 8–11
. During mitosis, in a process known as centrosome maturation12,13
, the PCM increases in size and γTuRCs are recruited from the cytosol, thereby promoting microtubule nucleation. While proteomic analyses have revealed hundreds of centrosomal components1, 14–18
, electron microscopy (EM) studies have only provided insights into the ultrastructural organization of centrioles5–7, 19–21
and basal bodies22–24
. By contrast, even with the use of electron tomography methods19,20,27,28
, no organizational pattern of PCM components has been discerned.
Studies on salt-stripped centrosomes have revealed a fibrous scaffold running throughout the PCM made of 12–15 nm fibers29,30
. Unfortunately, the molecular identity of these fibers has remained elusive and their role in organizing the PCM is unknown. Previous reports have also hypothesized the existence of a layer of proteins that attaches the PCM to the centriole, PCM tube9,31,32
. But if this layer exists, its components and function are unclear.
Here, we re-evaluate the notion that the PCM is an amorphous structure. Using a combination of three-dimensional (3D) Structured Illumination Microscopy (SIM), Stochastic Optical Reconstruction Microscopy (STORM)33–35
sub-diffraction resolution imaging methods and 3D image processing, we have begun to quantitatively map the architecture of the PCM. By examining the distribution and orientation of centrosomal proteins critical for centrosome maturation1,36
, we show that the PCM is composed of two major domains with distinct molecular composition and architecture. The PCM layer most proximal to the centriole wall is made of Plp fibrils that radiate outward from the centriole wall to the outer PCM matrix. RNAi experiments shows that Plp’s elongated structures provide a scaffold critical for the 3D organization of the outer PCM matrix.