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Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method

ABSTRACT: Centrosomes are small cytoplasmic organelles that play fundamental roles in a range of cellular processes. Nearly all vertebrate cell types contain centrosomes, but whether centrosome composition and function differ between cell types, tissues and pathologies remains an outstanding question. Until now, probing centrosome composition has relied on proteomic profiling of centrosomes obtained by consecutive sucrose density gradient centrifugations, requiring up to one billion cells, thus rendering studies with multiple conditions cumbersome. Here we describe centrosome affinity capture (CAPture)-mass spectrometry (MS), a method that achieves high-coverage proteomes from 20-30 million cells. Utilising a biotin-labelled peptide derived from the CCDC61 protein, CAPture isolates intact centrosomes in a manner dependent on Ninein, a conserved centrosome component. CAPture-MS performs well across several untransformed and primary cell lines, thereby enabling comparisons of high-resolution centrosome proteomes. Using distal appendage proteins as an example, we demonstrate the utility of CAPture-MS for dissecting hierarchical interactions within the centrosome. Overall CAPture-MS represents a powerful tool to unveil temporal, regulatory, cell type- and tissue-specific changes in centrosome proteomes in health and disease.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Spleen

SUBMITTER: Evangelia Papachristou

LAB HEAD: Fanni Gergely

PROVIDER: PXD040308 | Pride | 2023-10-18

REPOSITORIES: Pride

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			Action	DRS
	QE_HF_BEAD_ONLY.msf	Msf
	QE_HF_BEAD_ONLY.raw	Raw
	QE_HF_CENT_BO_1.raw	Raw
	QE_HF_CENT_PEP_1.raw	Raw
	QE_HF_CENT_PEP_2.raw	Raw

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Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method

Project description:Centrosomes are small cytoplasmic organelles that play fundamental roles in a range of cellular processes. Nearly all vertebrate cell types contain centrosomes, but whether centrosome composition and function differ between cell types, tissues and pathologies remains an outstanding question. Until now, probing centrosome composition has relied on proteomic profiling of centrosomes obtained by consecutive sucrose density gradient centrifugations, requiring up to one billion cells, thus rendering studies with multiple conditions cumbersome. Here we describe centrosome affinity capture (CAPture)-mass spectrometry (MS), a method that achieves high-coverage proteomes from 20-30 million cells. Utilising a biotin-labelled peptide derived from the CCDC61 protein, CAPture isolates intact centrosomes in a manner dependent on Ninein, a conserved centrosome component. CAPture-MS performs well across several untransformed and primary cell lines, thereby enabling comparisons of high-resolution centrosome proteomes. Using distal appendage proteins as an example, we demonstrate the utility of CAPture-MS for dissecting hierarchical interactions within the centrosome. Overall CAPture-MS represents a powerful tool to unveil temporal, regulatory, cell type- and tissue-specific changes in centrosome proteomes in health and disease.

2023-10-18 | PXD040309 | Pride

Proteomic profiling of centrosomes isolated from HAP1 cells

Project description:Centrosomes are cytoplasmic membraneless organelles that comprise a pair of centrioles, and a pericentriolar material. We developed a new centrosome affinity capture method termed CAPture that achieves high-coverage proteomes from 20-30 million cells. Here we present CAPture-MS data from HAP1 cells (WT and two CEP83-KO clones) demonstrating the power and sensitivity of CAPture-MS to detect centrosomal distal appendages hierarchical interactions.

2023-10-17 | PXD043692 | Pride

Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method

Project description:Centrosomes are the major microtubule-organising centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease aremajor unresolved questions, yet currently available centrosome isolation protocols are cumbersome, time-consuming and lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step method to obtain high-resolution centrosome proteomes from untransformed and primary cell lines. Utilising a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell typedependent heterogeneity in centrosome composition, dissect hierarchical interactions and identify novel components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell type- and tissue-specific changes in centrosome proteomes in health and disease.

2023-09-18 | PXD043906 | Pride

Gene expression in mitotic tissues of Drosophila larvae without centrosomes or too many centrosomes

2012-08-20 | GSE35240 | GEO

Gene expression in mitotic tissues of Drosophila larvae without centrosomes or too many centrosomes

Project description:Centrosome defects are a common feature of many cancers. Surprisingly, flies can proceed through the majority of development without centrosomes or with amplified centrosomes in most of their cells. It is unclear whether this is because centrosome defects do not cause many problems in Drosophila cells, or because they can adapt to cope with any problems that arise. Indeed, centrosome loss and centrosome amplification predispose fly brain cells to form tumours. Here we assess how centrosome loss or centrosome amplification perturbs cell physiology by profiling the global transcriptome of Drosophila larval brains and imaginal discs that either lack centrosomes or have too many centrosomes. Mitotic tissues (brains and imaginal discs) were dissected from 3rd instar Drosophila larvae of mutants lacking centrosomes (DSas-4 and DSas-6), a strain with too many centrosomes (SakOE) and two different wild type strains (w67 and OregonR). We extracted RNA from three biological replicates per strain and used it for hybridisation to Affymetrix Drosophila Genome 2.0 arrays. Per biological sample, material dissected from ten larvae was pooled. Gene expression of the mutant strains was compared to both wild type controls.

2012-08-20 | E-GEOD-35240 | biostudies-arrayexpress

Proteomic analysis of centrosome-associated proteins between non-activated non-polarized and activated polarized murine B cells R#1 (R#2 doi: 10.1038/ncomms10969) & R#3

Project description:Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative proteomics on purified centrosomes from resting, non-activated non-polarized, and activated polarized mouse B cells, we established a list of proteins differentially associated with the centrosome between resting and polarized states.

2022-12-12 | PXD038725 | Pride

Gene expression comparison of MCF10A cells with and without extra centrosomes through overexpression of PLK4

Project description:The presence of extra centrosomes is a feature of human tumours. However, it is unclear what are the changes elicited by the presence of these abnormalities. To determine the changes in gene expression induced by centrosome amplification, human mammary epithelial cells, MCF10A, expressing a doxycycline inducible PLK4 cDNA were used. Centrosome amplification was induced for 48 hrs upon the addition of doxycyclin to the culture medium. RNA was purified from MCF10A cells without extra centrosomes (no dox) and with extra centrosomes (dox) and processed for microarray analysis.

2018-02-10 | E-MTAB-6415 | biostudies-arrayexpress

Oncogene-like induction of cellular invasion from centrosome amplification

Project description:Centrosome amplification has long been recognized as a feature of human tumors, however its role in tumorigenesis remains unclear. Centrosome amplification is poorly tolerated by non-transformed cells, and, in the absence of selection, extra centrosomes are spontaneously lost. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumors, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumor progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behavior is similar to that induced by overexpression of the breast cancer oncogene ErbB2 and indeed enhances invasiveness triggered by ErbB2. We show that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation. genome-wide human SNP 6.0 arrays from Affymetrix was used to determine the copy number changes of MCF10A cells with extra centrosomes or depleted of MCAK after grown 4 days in 3-D cultures

2014-03-01 | E-GEOD-55042 | biostudies-arrayexpress

Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals

Project description:Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of human cancer remains unclear. Here, we report the creation of a mouse model in which centrosome number can be persistently increased in the absence of additional genetic defects. We show that extra centrosomes increase tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive development of spontaneous tumors in multiple tissues. Tumors with centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship between centrosome amplification, genomic instability and tumor development. The sequences in this dataset result from random whole-genome DNA sequencing of spontaneous T- cell lymphomas, B-cell lymphomas, squamous cell carcinomas and one sarcoma from doxycycline-treated Plk4 mice.

2017-02-04 | E-MTAB-5043 | biostudies-arrayexpress