Tackling centrosome biology through gene targeting in chicken B cells

Chavali, Pavithra L.; Gergely, Fanni

doi:10.1016/bs.mcb.2015.03.008

Cited by 6 publications

(6 citation statements)

References 31 publications

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“…In sucrose density gradient centrifugation, PCM1‐GFP and the centriolar satellite‐associated ubiquitin ligase MIB1 were found in fractions 30–70% (Fig H). Centrosomes are expected to sediment in the higher sucrose fractions (Chavali & Gergely, ); indeed, the 60–70% fractions of WT cells appeared more enriched for the centriolar protein centrin 2 than the equivalent fractions in the acentriolar cells. To minimise co‐isolation of centrosomes, only fractions 30–50% were pooled for subsequent GFP and control IgG immunoprecipitations (Fig H).…”

Section: Resultsmentioning

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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Section: Resultsmentioning

confidence: 99%

Centriolar satellites are acentriolar assemblies of centrosomal proteins

et al. 2019

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“…Using gene targeting we created chicken DT40 cell lines in which either HSET or the HBR of CEP215 was disrupted. The HSET knockout line (HSET KO ) was generated by replacing the exons encoding the tail and stalk domains (aa1–345) with antibiotic resistance genes 38 ( Supplementary Fig. 5a ).…”

Section: Resultsmentioning

confidence: 99%

“…Gene targeting was performed according to standard protocol 38 . Briefly, homology arms were cloned into pJET or PGEMT-Easy and subcloned into pBluescript II SK − (pSK).…”

Section: Methodsmentioning

confidence: 99%

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A CEP215–HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

et al. 2016

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Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215–HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome–spindle pole interface by the CEP215–HSET complex could promote survival of cancer cells containing supernumerary centrosomes.

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“…These cells are unusual as they exhibit higher frequencies of targeted versus random integration of transgenes into the genome, in comparison to other somatic vertebrate cell lines (Buerstedde and Takeda 1991 ). For this reason, these cells have been particularly useful for loss-of-function and protein-tagging in vitro studies (Chavali and Gergely 2015 ; Kobayashi et al 2015 ; Daly et al 2016 ).…”

Section: Gene Editing Of Avian Somatic Tissue and Cellsmentioning

confidence: 99%

Gene editing in birds takes flight

2017

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The application of gene editing (GE) technology to create precise changes to the genome of bird species will provide new and exciting opportunities for the biomedical, agricultural and biotechnology industries, as well as providing new approaches for producing research models. Recent advances in modifying both the somatic and germ cell lineages in chicken indicate that this species, and conceivably soon other avian species, has joined a growing number of model organisms in the gene editing revolution.

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Tackling centrosome biology through gene targeting in chicken B cells

Cited by 6 publications

References 31 publications

Centriolar satellites are acentriolar assemblies of centrosomal proteins

Centriolar satellites are acentriolar assemblies of centrosomal proteins

A CEP215–HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

Gene editing in birds takes flight

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