Flj23047

One emerging idea is that the centrosome functions not only as a MT-organizing center but also as a solid-state platform for regulatory molecules and a junction for signaling processes [9, 27-29] (see Chapter 8). From this perspective, it would seem plausible that a complex structural scaffold may be required to allow for the specific and orderly binding of numerous factors. The many coiled-coil proteins may therefore act as a core structure to which weakly interacting proteins can bind transiently.

Another possible explanation for the multitude of centrosomal coiled-coil proteins is that interactions between such proteins may be ideally suited to confer the striking flexibility that recent experiments have brought to light [30]. Live cell imaging performed on interphase centrosomes has in fact revealed that the two centrioles within a given centrosome display a much more dynamic behavior than had previously been appreciated. This plasticity may require a dynamic archi-tecure that a rigid matrix, composed of only few components, would be less likely to provide. Similarly, it is possible that numerous structural components are required to generate a sufficiently malleable and responsive structure to allow for cell cycle- or differentiation-dependent alterations in centrosome structure and composition.

Two proteins identified in the survey, termed ALMS1 and OFD1, have previously been identified genetically as being linked to human diseases. In particular, the C-terminal half of ALMS1 was localized to the centrosome by tagging [21] and, independently, antibodies to OFD1 have been shown to decorate the centrosome [31]. Both proteins need further investigation to confirm their association with the cen-trosome but their identification as candidate centrosome components is intriguing. The diseases caused by defects in these genes are relatively rare and poorly understood. Patients with Alstrom syndrome (ALMS1) display a complex set of symptoms. Childhood obesity starts at the early age of 6 months and many patients develop type 2 diabetes. The disease is also associated with neurosensory defects and subsets of patients show dilated cardiomyopathy, hepatic dysfunction, hypothyroidism, male hypogonadism, short stature and mild developmental delay [32, 33]. The symptoms of oral-facial-digital syndrome type I (OFD1) are more straightforward. Typically, the patients have malformations of the digits and face or oral cavity such as a cleft palate. In addition, two-fifths of patients have defects in the central nervous system [34]. Thus, in both of these diseases, a considerable variety of tissues appears to be affected. With the identification of the mutated proteins as putative centrosome components, it is tempting to speculate that the cause of these diseases is disruption of centrosome (or basal body) function during development. This adds to emerging evidence suggesting a critical role of the centrosome/basal body in forming the architecture of particular tissues [35, 36]. Perhaps we are about to witness the dawn of "centrosomopathies".

7.5 Cell Cycle Changes In Centrosome Composition | 135 Cell Cycle Changes in Centrosome Composition

The structure and behavior of the centrosome changes markedly throughout the cell cycle. This is reflected by the appearance or disappearance of individual centro-somal proteins at particular cell cycle stages, notably at the G2/M transition when the centrosome undergoes maturation in preparation for spindle formation [37, 38]. Other potentially important changes concern the establishment and dissolution of different types of linkages that are thought to connect the two parental cen-trioles to each other, and each parental centriole (mother centriole) to its growing pro-centriole (daughter centriole), respectively (Figure 7.3A). Critical changes undoubtedly occur also during duplication of the centrioles in S phase, and it has long been known that the two parental centrioles can be distinguished from each other by the cell cycle-regulated appearance of appendages at the distal and sub-distal end of only the older ("mature") centriole [39]. The functions of these appendages remain to be fully understood, but roles in microtubule anchoring and the formation of the primary cilium have been documented [39].

Cell cycle-dependent changes in centrosome structure have been extensively described at the electron microscopic level, but their detection by light microscopy remains difficult. This is a reflection of both the lower resolution of light microscopy and the internal organization of the centrosome. Antibodies against most PCM components (including y-tubulin) will stain material associated with both centrioles and, therefore, give rise to two closely spaced dots under the microscope. Depending on the plane of focus and the spatial orientation of the organelle, however, the two dots will occasionally coalesce into one. As cells progress through the cycle, the two-dot staining pattern will not visibly change, even though the two cen-trioles duplicate (Figure 7.3B). This is because parental centrioles and their closely apposed pro-centrioles are not easily visualized as separate entities by antibodies directed against PCM components.

A further complication arises from the fact that in some G1 phase cells the two parental centrioles will be very close to each other (thus difficult to recognize as two distinct dots), whereas in others they split over distances of several microns (hence clearly producing two dots) (e.g. [40, 41]). Unfortunately, these split single centro-somes are frequently - but erroneously - considered as two already duplicated centrosomes (Figure 7.3C). Because of the small size of centrioles, the scarcity of centriolar markers and the geometric considerations described above, a rigorous and reliable quantitative analysis of centrosome duplication by light microscopy remains a difficult task.

Using cell lines stably expressing GFP-centrin and high resolution microscopy, Bornens and colleagues have been able to visualize both centrioles and growing pro-centrioles [30, 42]. By carefully studying centrosome duplication in S phase cells, these workers were able to observe two faint dots (representing pro-cen-trioles) appearing next to the two more intensely labeled dots (representing the parental centrioles). Subsequently, the brightness of the pro-centriolar signals increased during S phase progression, so that by G2, four dots could be seen (repre-

senting two doublets of duplicated centrioles). Thus, by studying centriolar markers under optimal conditions, it is possible to monitor centrosome duplication by observing the increase in the number of fluorescently labeled dots from two to four. However, as signals frequently coalesce, depending on geometry and plane of focus, the unequivocal counting of these signals is far from trivial.

m Figure 7.3 Cell cycle dynamics of the centrosome. This figure summarizes, in schematic form, a few considerations that are relevant to the study of centrosome dynamics by light microscopy. (A) Two different centriolar linkers probably exist in the centrosome, one linking the emerging or newly formed (daughter) centriole to the parental centriole, the other linking two parental cen-trioles that will themselves give rise to pro-centrioles during S phase. The latter connection must be broken during G2 to allow the two duplicated centrosomes to separate for spindle formation. The former link must be dissolved at the end of mitosis to allow the two centrioles to move separately and, most likely, to allow subsequent duplication. (B) The limits of resolution of light microscopy and the broad distribution of many pericentriolar matrix proteins make it difficult to monitor centrosome duplication by fluorescence microscopy. (C) The different possible orientations of multiple centrioles relative to each other and the observer and the ability of the centrosome to split (because of centrioles separating from each other) constitute further potential sources of confusion when counting centrosomes.

The Impact of MS on Centrosome Analysis during Cell Cycle and Development

We are confident that the ability to analyse the centrosome by mass spectrometry will greatly help in monitoring changes occurring at this organelle as cells proliferate and differentiate. In particular, it should be possible to purify centrosomes from cells synchronized at different stages of the cell cycle. With increasing sensitivity of mass spectrometry and a concomitant decrease in the amount of material needed for analysis, it will also become attractive to apply mass spectrometry to study changes in centrosome composition during the development of different tissues. A comparison of the components in such preparations should theoretically reveal proteins that are added or lost during the cell cycle and/or during differentiation. An important advance favoring this type of study is the emerging ability to use non-radioactive heavy isotopes for protein labeling [43-46]. Since the masses of peptides derived from isotope-labeled cells will be shifted relative to those from un-labeled cells, it is possible to carry out quantitative comparisons of protein levels between two different cell populations by mass spectrometry. Particularly powerful are experimental protocols in which labeled and unlabeled samples are mixed prior to organelle purification and mass spectrometric analysis, eliminating errors due to variations in sample processing.

Another, wide open field for future investigation concerns the role ofposttransla-tional modifications, notably phosphorylation, in the control of centrosome structure and activity [47]. Clearly, the increasing sensitivity of mass spectrometry and the continued development of peptide fractionation and isolation procedures [48-51] hold great promise for elucidating these regulatory events. In the case of the centrosome, these approaches appear particularly important and attractive, as conventional biochemical approaches for studying posttranslational modifications are severely limited by the low amounts of centrosomal proteins that can be prepared for study.

138 | 7 A Proteomic Approach to the Inventory of the Human Centrosome 7.7

Expanding Proteomic Information into Knowledge about Function

The described proteomic approach has provided an invaluable source of information on the component parts of the human centrosome [21], and we expect that mass spectrometry will continue to provide insight into cell-cycle and developmental changes in centrosome composition. Clearly, though, the persisting key task is to use this information to derive models of how the whole centrosome works. In the past, centrosome proteins have often been discovered through genetic screens or biochemical searches for interacting partners of already characterized proteins. And in at least some of these cases, clues to the functions of newly discovered proteins were thus available. For proteins discovered through proteomics, with no obvious functional links or insight from sequence information, knowing where to start to decipher a function is less obvious. A priori, overexpression of a novel protein may lead to observations (e. g. y-tubulin recruitment) that suggest a specific function (e.g. [12]). Conversely, depletion of a protein, notably by siRNA technology, may yield information on a loss-of-function or hypomorphic phenotype [52, 53]. However, by siRNA it may be difficult to deplete some centrosomal proteins sufficiently to produce a clear-cut phenotype. In particular, in cases where large cytoplasmic pools exist and turnover at the centrosome occurs, very extensive depletion may be required before the function of the centrosomal pool is impaired. Thus, in spite of the undisputed power of siRNA approaches, it will ultimately be important to assess the function of selected centrosomal proteins by gene knock-out strategies. Finally, another persisting challenge is to develop novel assays to probe various aspects of centrosome function. These assays should focus not only on the roles of centrosomes in microtubule organization, cell polarity and motility, but also in cell cycle progression and development.

Conclusion and Prospects

The use of proteomic approaches to investigate the centrosome has been remarkably successful. It thus seems legitimate to hope for a comprehensive description of centrosome composition in a not-too-distant future. Furthermore, mass spectro-metry holds great promise for monitoring changes in centrosome function. This is true regardless of whether changes in centrosome behavior during cell cycle progression or differentiation involve changes in protein composition or in the activity of particular components in response to posttranslational modifications. As in many other fields to which mass spectrometry has been applied as an analytical technique, it is thus likely to develop into a core tool for centrosome research.

Acknowledgments

We thank Thibault Mayor for his centrosome purifications in the early stages of the project, Peter Mortensen for his important contribution to the development of PCP, Monika Matzner for expert assistance with the tagging of candidate proteins, Roman K├Ârner for critical reading of the manuscript and many laboratory members for helpful discussions. The work in the authors' laboratory was generously supported by the Max-Planck-Society (CJW and EAN) and the Danish National Research Foundation (JSA and MM).

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