GFP-centrin as a marker for centriole dynamics in living cells

White, R. A. S.; Pan, Zuanfeng; Salisbury, Jeffrey L.

doi:10.1002/(sici)1097-0029(20000601)49:5<451::aid-jemt7>3.0.co;2-9

Cited by 62 publications

(45 citation statements)

References 35 publications

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“…As already shown in other mammalian cells, including HeLa and Syrian hamster cells (White et al, 2000;Piel et al, 2001), we found that constitutive expression of exogenous centrin2 does not interfere with CHO cell growth. Consequently, we were able to establish stable cell lines expressing GFP-centrin that allowed us to directly monitor the process of centrosome replication during HU treatment.…”

Section: Discussionsupporting

confidence: 86%

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Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2

Kuriyama

Terada

Lee

et al. 2007

Journal of Cell Science

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Centrosome duplication is tightly coupled with the cell cycle and neither too many nor too few centrosomes are induced in a normal cell. To study how centrosome assembly is regulated, we analyzed the abnormal process of multiple centrosome replications in Chinese hamster ovary (CHO) cells induced by hydroxyurea (HU), which is known to uncouple the centrosome cycle from the cell cycle. Green fluorescent protein (GFP)-tagged centrin2 expressed in CHO cells labels both centrioles and the pericentriolar material (PCM). Counting fluorescent spots of GFP-centrin in synchronized cells showed that in G1/S-arrested cells, centrioles are initially duplicated in a template manner. Further treatment with HU overrides the suppression of excess centriole/centrosome replication in a cell where the full complement of centrioles/centrosomes already exists. Time-lapse fluorescence microscopy revealed that small centrin-containing foci emerged in the cytoplasm during HU treatment. These foci are surrounded by a PCM cloud and their number continuously increases as cells are exposed to HU for longer periods of time. Both the centrosome and cytoplasmic foci are highly mobile, continuously changing their position in a manner dependent on microtubules/microtubule dynamics. The centrosome number increases as small foci grow in size and resolve into recognizable centrosomes. As this occurs in a random fashion, the cells arrested longer with HU induced highly heterogeneous numbers of centrosomes.

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Section: Discussionsupporting

confidence: 86%

“…1D-F), with varying distances between them. As centrin is a widely used centriolar marker (White et al, 2000;Piel et al, 2001), they probably correspond to the centrioles of the centrosomes. The majority of cells contain dots of more or less equal size.…”

Section: Centrosomes Are Labeled With Gfp-tagged Centrin In Cho Cellsmentioning

confidence: 99%

Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2

Kuriyama

Terada

Lee

et al. 2007

Journal of Cell Science

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“…The centrosome localization of centrin and Plk2 have been shown previously. 9,10 SEPT7 is one of the septin family members, a highly conserved GTP-binding protein originally identified in a genetic screening of S. cerevisiae mutants defective in cytokinesis, and is known to play roles in cytokinesis, membrane trafficking and as structural scaffolds. 11,12 Although centrosome localization of SEPT7 had not been reported previously, our data show that SEPT7 localizes to centrosomes.…”

Section: Resultsmentioning

confidence: 99%

Suppression of centrosome duplication and amplification by deacetylases

et al. 2012

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“…Use of GFPtagged centrin along with EM studies has demonstrated that the two centrioles within an interphase centrosome appear and behave quite differently with respect to size and colocalization with MTOC [39,55,76], and this difference of centrioles marked by GFP-centrin is commonly used to help define the localization of centrosomal associated proteins [39,55,76]. The two centrioles of a centrosome are thought to carry distinct activities, with the larger mature "mother" centriole (MC) capturing and anchoring nucleated MTs and providing important signals to the midbody that promote cytokinesis [39,76,[94][95][96].…”

Section: Discussionmentioning

confidence: 99%

“…The YFP-tubulin expression construct was purchased from Clonetech (Palo Alto, CA). Dr. Sean Munro (MRC, Cambridge, England) kindly provided the expression vectors encoding the RFP-pericentrin PACT domain, the GFP fused AKAP450 C-terminal region [54], and the GFP-tagged centrin expression construct [55] was a gift of Dr. Jeffrey Salisbury (Mayo Clinic, Rochester, MN)…”

Section: Expression Constructs and Transient Transfection Of Cellsmentioning

confidence: 99%

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

Don

Dallapiazza

Bennin

et al. 2006

Experimental Cell Research

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Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition, and the formation of aberrant nuclei [1]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT), and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells, and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome, resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs, and a p53 dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFPcentrin tagged centrioles, the mature centriole present at microtubule foci, indicate that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology, and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities.

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GFP-centrin as a marker for centriole dynamics in living cells

Cited by 62 publications

References 35 publications

Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2

Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2

Suppression of centrosome duplication and amplification by deacetylases

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

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