Lis Jakobsen

Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods

Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate key signalling events. Despite recent advances, the molecular basis for the plethora of processes coordinated by centrosomes is not fully understood. We have combined protein identification and localization, using PCP‐SILAC mass spectrometry, BAC transgeneOmics, and antibodies to define the constituents of human centrosomes. From a background of non‐specific proteins, we distinguished 126 known and 40 candidate centrosomal proteins, of which 22 were confirmed as novel components. An antibody screen covering 4000 genes revealed an additional 113 candidates. We illustrate the power of our methods by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads to the further functional characterization of centrosomal proteins.

Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling

The ability to purify cell organelles and protein complexes on a large scale, combined with advances in protein identification using mass spectrometry, has provided a wealth of information regarding protein localization and function. A major challenge in these studies has been the ability to identify bona fide organelle components from a background of co-purifying contaminants because none of the available biochemical purification protocols afford pure preparations. Since this situation is unlikely to change alternative strategies have been devised to meet this challenge by making use of the information inherent in the fractionation profile of organelles isolated by density gradient centrifugation. In this chapter we describe strategies based on protein correlation profiling and quantitative mass spectrometry to sort out likely candidates. The organelle inventories defined by these methods are suitable to guide future functional experiments.

Centrosome Isolation and Analysis by Mass Spectrometry-Based Proteomics

Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined with advances in protein identification using mass spectrometry-based proteomics, have revealed multiple centriole-associated proteins that are conserved during evolution in eukaryotes. Despite these advances, the molecular basis for the plethora of processes coordinated by cilia and centrosomes is not fully understood. Considering the complexity and dynamics of centriole-related proteomes and the first-pass analyses reported so far, it is likely that further insight might come from more thorough proteome analyses under various cellular and physiological conditions. To this end, we here describe methods to isolate centrosomes from human cells and strategies to selectively identify and study the properties of the associated proteins using quantitative mass spectrometry-based proteomics.

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-β1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.

Identification and characterization of two novel centriolar appendage component proteins

The mother centriole forms the basis of the primary cilium. As the cilium assembles, the mother centriole matures and differentiates into the basal body, and a number of fibrous structures are formed that add to the complexity, including the distal and subdistal appendages. A number of the proteins corresponding to these structures are identified already, but given the complexity of the basal body, the macromolecular composition of some of these appendages remain unknown. To date, the proteins ninein and Cep170 are believed to be a part of the subdistal appendages, and Cep164, outer dense fiber 2, ODF2/cenexin, Cep290, Ofd1 compose the distal appendages. Most of these appendage structures have been reported to play a role for cilia assembly. We previously characterized the centrosome proteome of human lymphoblastic KE-37 cells using quantitative mass spectrometry, which identified 40 novel candidate proteins (Jakobsen et al., 2011). Using immunoflourescence- and immunogold electron microscopy on different human culture cells we identified proteins localizing asymmetrically to the centrioles, and two of these appeared to be new appendage proteins, one distal- (Cep37) and one subdistal (Cep128). Interestingly, in addition Cep37 also localize to the ciliary tip and more faintly along the axoneme. Preliminary results indicate that depletion of Cep37 reduces length of cilia in RPE cells. Cep128 does not affect RPE or hFF cells ability to form primary cilia, but they do show a higher number of pericentriolar dense bodies or satellites as well as a higher number of non exocytotic vesicles in line with the cilium.

Identification and functional analysis of novel centrosomal proteins to study their implication in human disease

The physiological importance of cilia is underscored by the ever-growing list of ‘ciliopathies’ associated with ciliary dysfunction. The many overlapping phenotypes of these syndromes might reflect the complexity of the ciliary proteome and the numerous functions of cilia. Likewise, dysfunction of a number of centrosomal proteins has been linked to human diseases. As centrosomes and cilia are closely linked, centrosomal abnormalities can also cause disease by affecting cilia formation, for example by affecting the docking of the basal body to the cell membrane, transport processes relying on microtubule interactions with the centrosome, targeting of membrane vesicles or the intraflagellar transport required for cilia assembly. It might thus be that a number of diseases could be more correctly referred to as ‘centrosomeopathies’. Despite the identification of many centrosomal and ciliary proteins, it is still not known how many of these components interact and function. We have recently characterized the human centrosome proteome in depth using a quantitative mass spectrometry-based method in combination with an antibody-based screen, identifying several novel and candidate centrosomal proteins. The thereby obtained human centrosome proteome provides an excellent basis for further experiments to elucidate the multiple functions of the centrosome, including its role in human disease such as ciliopathies and cancer development. We are currently undertaking more functional experiments based on the improved centrosome proteome to investigate a possible role of these proteins in processes such as centriole duplication and ciliogenesis, both of which are processes with links to human disease.