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Figure 7.1 The centrosome in perspective. The size of the centrosome compared to other cellular organelles in the cell is shown to scale, together with estimates of their abundance.

What is a Centrosome Component?

The absence of a clearly visible (membranous) boundary raises the general question of how to define a centrosomal component. With our limited current knowledge of centrosome structure it is in fact difficult to determine exactly where this organelle ends. Boundaries are further blurred by the fact that many components, although clearly enriched at the centrosome, also occur in large cytoplasmic pools. Prominent examples for proteins existing both at the centrosome and in the cytoplasm include y-tubulin and centrin [7, 8]. Nevertheless, these proteins can readily be used as markers in fluorescence microscopy, because their enrichment at the centrosome results in a high signal density. Many microtubule (MT)-associated proteins are also expected to be present in relatively high concentrations at the cen-trosome. To distinguish such proteins from the genuine (core) components, cells are usually exposed to either cold or nocodazole treatment, both of which depoly-merize MTs. Those proteins whose centrosome localization resists cold or nocado-zole treatment are operationally defined as core centrosome components.

Of particular importance is the highly dynamic structure of the centrosome which displays major structural alterations and compositional changes during the cell cycle [9, 10] (see also Chapter 9). Several proteins have been described whose centrosomal localization is severely diminished at the onset of mitosis e. g. PCM-1, C-Nap1, and Nlp [11-13] while others, notably the y-tubulin ring complex, are recruited in greater quantity as the centrosome undergoes so-called maturation at the G2/M transition [14]. Yet other proteins localize to the spindle poles only during mitosis, some of which, like NuMA [15], are nuclear proteins during interphase. In these latter cases, electron microscopy may be required to determine the exact location of such proteins in the area of the poles. As exempli fied by plants, spindle poles can clearly exist in the absence of centrosomes. Thus, although the terms "spindle pole" and "(mitotic) centrosome" are often used interchangeably, they do not necessarily describe the exact same structure. Thus, some proteins may accumulate in the vicinity of the poles without being components of the centrosome proper.

From a technical perspective, there are a number of issues that are becoming increasingly important the more widely the centrosome is being studied. In particular, it is important to bear in mind that animal sera occasionally contain antibodies with anti-centrosome activity even prior to immunization [16]. To make sure that a newly raised antibody does not erroneously report on centrosome association of a protein under study, it is critical, therefore, to perform control experiments with pre-immune rather than non-immune serum, i. e. serum collected from the same animal that was subsequently used for immunization rather than another individual of the same species. Furthermore, it is generally useful to confirm the im-munocytochemical localization of a novel endogenous protein by showing that an epitope-tagged product of the corresponding cDNA also localizes to the centrosome. However, such experiments do not necessarily constitute an infallible method either. In addition to the pitfall that overexpression might produce artefac-tual associations, we have occasionally (albeit rarely) observed that different tags yielded different results (e.g. [17], C. J. Wilkinson and E. A. Nigg, unpublished results). This may reflect misfolding or masking of targeting signals caused by particular tags. As with all (immuno-)cytochemical approaches, complementary biochemical data are thus desirable. This is true in spite of the fact that none of the available purification methods affords centrosomes without significant contamination by cellular proteins, so that biochemical approaches provide corroborating rather than unequivocal evidence.

Composition of the Human Centrosome: A Proteomic Approach

The exact number of centrosomal proteins is difficult to establish with confidence. Nevertheless, a survey of the literature suggests that nearly a 100 proteins associate with centrosomes at some stage of the cell cycle in different species. Of these, about 60 are expected to be present on the interphase centrosome in humans. Many centrosomal proteins have been discovered through genetic analyses or through the production of antibodies to purified centrosomes (or MT-binding proteins). The search for interaction partners of centrosomal components by yeast two-hybrid screens and immunoprecipitation experiments has further expanded the catalog. Most recently, mass spectrometry has been used to determine the composition of the y-tubulin ring complex [18]. Of great promise is the application of this technology to whole centrosomes which is now becoming feasible [19-21], as described in more detail below.

A priori, it might seem desirable to improve current protocols for centrosome purification, prior to subjecting such samples to a proteomic analysis. However, as the centrosome lacks a clear boundary, harsher purification methods inevitably entail the danger of losing peripheral components. In a recent study [21], we therefore decided to analyze a partially pure sample of centrosomes, taking advantage of both the increased sensitivity of contemporary mass-spectrometry instruments and advanced data analysis algorithms. In brief, two methods were used to generate peptides for mass spectrometric analysis. In one protocol, whole centrosome preparations were digested with trypsin, in the other, samples were first subjected to one-dimensional gel electrophoresis, followed by in-gel trypsin digestion of 15 slices. Peptides were then separated by nano-liquid chromatography before injection into a quadrupole time-of-flight mass spectrometer by an electrospray source.

This analysis identified over 2000 peptides corresponding to 500 proteins [21]. Since the centrosomes had been purified from asynchronously growing cells (with about 70% in G1, the remainder in S, G2 and M), we expected to find primarily, proteins associated with the interphase centrosome. Of the approximately 60 described components of the interphase centrosome, 47 were indeed found (Table 7.1). These included both prominent structural proteins (e.g. pericentrin,

Table 7.1 Known centrosome components detected by mass-spectrometric analysis of purified centrosomes*.

Structural components

Alpha tubulin Beta tubulin Gamma tubulin

Gamma-tubulin complex component 2 Gamma-tubulin complex component 3 Gamma-tubulin complex component 4, h76p Gamma-tubulin complex component 5 Gamma-tubulin complex component 6 Centrin 2 Centrin 3

AKAP450 (AKAP350, GC-Nap) Pericentrin/Kendrin

(alternatively spliced proteins) Ninein

Pericentriolar material 1 (PCM-1) ch-TOG protein C-Nap1, Cep250, Cep2 Centriole associated protein CEP110, Cepl, centriolin

Centrosomal P4.1-associated protein (CPAP)

CLIP-associating proteins CLASP1 and CLASP2

ODF2, cenexin

Lisi

Nudel

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