Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Bauer, Manuel; Cubizolles, Fabien; Schmidt, Alexander; Nigg, Erich A.

doi:10.15252/embj.201694462

Cited by 83 publications

(98 citation statements)

References 132 publications

(210 reference statements)

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“…For example, we observed over 2,100 blinking events in the upper centriole in Fig. 2C, whereas the copy number of CEP135 per centriole has been estimated to be 300-500 (23). To compare the photon counts of mCherry with well-established photoconvertible fluorophores, we created tubulin constructs fused to mEos3.2 (24) or mMaple3 (25).…”

Section: Resultsmentioning

confidence: 99%

Efficient switching of mCherry fluorescence using chemical caging

Cloin

Zitter

Salas

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantummechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores. fluorescent proteins | mCherry | localization microscopy | β-mercaptoethanol | photoactivation

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Section: Resultsmentioning

confidence: 99%

Efficient switching of mCherry fluorescence using chemical caging

Cloin

Zitter

Salas

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Although several factors involved in centriole biogenesis are among these 82 proteins, the three critical proteins that drive centriole formation, SAS‐6, STIL and CEP152 along with PLK4 were absent from CS‐WT (Banterle & Gonczy, ; Nigg & Holland, ). The low detection rates may simply reflect low abundance of these proteins, which is especially relevant because STIL and PLK4 are among the five least abundant proteins at the centrosome according to a recent study (Bauer et al , ). Ranking proteins based on their estimated centrosomal copy numbers revealed that the presence of centrosomal proteins in our filtered dataset and CS‐WT (nomenclature as in Fig A; Fig EV6 and Table EV4) is associated with higher centrosomal abundance.…”

Section: Discussionmentioning

confidence: 99%

Centriolar satellites are acentriolar assemblies of centrosomal proteins

et al. 2019

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Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)‐based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions. We defined an interactome consisting of 223 proteins, which showed striking enrichment in centrosome components. The proteome also contained new structural and regulatory factors with roles in ciliogenesis. Quantitative MS on whole‐cell and centriolar satellite proteomes of acentriolar cells was performed to reveal dependencies of satellite composition on intact centrosomes. Although most components remained associated with PCM1 in acentriolar cells, reduced cytoplasmic and satellite levels were observed for a subset of centrosomal proteins. These results demonstrate that centriolar satellites and centrosomes form independently but share a substantial fraction of their proteomes. Dynamic exchange of proteins between these organelles could facilitate their adaptation to changing cellular environments during development, stress response and tissue homeostasis.

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“…Centrosomes participate in mitotic spindle formation, and thereby ensure faithful chromosome segregation during cell division. Failures in centriole duplication result in abnormal centriole numbers, which have been linked to genomic instability and tumorigenesis 28,2930,31 Wilms tumors32 and mantle cell lymphoma 33.…”

Section: Discussionmentioning

confidence: 99%

Analysis of potential genes associated with primary cilia in bladder cancer

Du¹,

Lü²,

Sheng³

et al. 2018

CMAR

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BackgroundDysfunction of primary cilia (PC), which could influence cell cycle and modulate cilia-related signaling transduction, has been reported in several cancers. However, there is no evidence of their function in bladder cancer (BLCA). This study was performed to investigate the presence of PC in BLCA and to explore the potential molecular mechanisms underlying the PC in BLCA.Patients and methodsThe presence of PC was assessed in BLCA and adjacent non-cancerous tissues. The gene expression dataset GSE52519 was employed to obtain differentially expressed genes (DEGs) associated with PC. The mRNA expression of the DEGs were confirmed by Gene Expression Profiling Interactive Analysis. The DEGs properties and pathways were analyzed by Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Genomatix software was used to predict putative transcription factor binding sites (TFBS) in the promoter region of DEGs, and the transcription factors were achieved according to the shared TFBS, which were supported by the ChIP-Sequence data.ResultsPC were found to be reduced in BLCA tissue samples in this study. Seven DEGs were observed to be associated with PC, and gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that these DEGs exhibited the properties and functions of PC, and that the Hedgehog signaling pathway probably participated in the pathogenesis and progression of BLCA. The mRNA expression of the seven DEGs in 404 BLCA and 28 normal tissue samples were analyzed, and five DEGs including CENPF, STIL, AURKA, STK39 and OSR1 were identified. Five TFBS including CREB, E2FF, EBOX, ETSF and HOXF in the promoter region of five DEGs were calculated and the transcription factors were obtained according to the shared TFBS.ConclusionPC were found to be reduced in BLCA, and the potential molecular mechanisms of PC in BLCA helped to provide novel diagnosis and therapeutic targets for BLCA.

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Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Cited by 83 publications

References 132 publications

Efficient switching of mCherry fluorescence using chemical caging

Efficient switching of mCherry fluorescence using chemical caging

Centriolar satellites are acentriolar assemblies of centrosomal proteins

Analysis of potential genes associated with primary cilia in bladder cancer

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