Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

Keppler, Antje; Kindermann, Maik; Gendreizig, Susanne; Pick, Horst; Vogel, Horst; Johnsson, Kai

doi:10.1016/j.ymeth.2003.10.007

Cited by 185 publications

(152 citation statements)

References 20 publications

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“…SNAP is derived from alkyl guanine DNA alkyl transferase, a DNA repair enzyme that irreversibly transfers an alkyl group from its substrate to a reactive cysteine residue (32)(33)(34)(35)(36). The availability of cell-permeable nonfluorescent and fluorescent SNAP substrates, such as TMR-Star, allows for a broad variety of applications.…”

Section: Significancementioning

confidence: 99%

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Hoboth

Müller

Ivanova

et al. 2015

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

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Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin.

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Section: Significancementioning

confidence: 99%

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Hoboth

Müller

Ivanova

et al. 2015

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

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“…1 A). The SNAP tag, a modified form of the enzyme O 6 -alkylguanine-DNA alkyltransferase, allows temporally and spatially defined cohorts of proteins to be selectively labeled and detected (Juillerat et al, 2003;Keppler et al, 2004). The tag binds covalently to O 6 -benzylguanine (BG), resulting in the irreversible transfer of the substituted benzyl group to the reactive thiol within the SNAP tag.…”

Section: Resultsmentioning

confidence: 99%

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

Stoops¹,

Hull²,

Olesen³

et al. 2015

J Gen Physiol

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“…For additional confirmation, we generated stably transformed S2R+ cells expressing SNAPf-tagged Cid. This particular Cid variant can be fluorescently labeled by addition of TMR-Star, a tetramethylRhodamine derivative, to the culture medium (Keppler et al, 2004). Quantification after labeling with such a low molecular weight compound appeared preferable to anti-Cid immunolabeling where complications with variable epitope accessibility during different cell cycle stages were encountered.…”

Section: The Total Amount Of Centromeric Cid Increases During G1mentioning

confidence: 99%

Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells

Lidsky

Sprenger

Lehner

2013

Journal of Cell Science

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SummaryCentromeres are specified epigenetically in animal cells. Therefore, faithful chromosome inheritance requires accurate maintenance of epigenetic centromere marks during progression through the cell cycle. Clarification of the mechanisms that control centromere protein behavior during the cell cycle should profit from the relatively simple protein composition of Drosophila centromeres. Thus we have analyzed the dynamics of the three key players Cid/Cenp-A, Cenp-C and Cal1 in S2R+ cells using quantitative microscopy and fluorescence recovery after photobleaching, in combination with novel fluorescent cell cycle markers. As revealed by the observed protein abundances and mobilities, centromeres proceed through at least five distinct states during the cell cycle, distinguished in part by unexpected Cid behavior. In addition to the predominant Cid loading onto centromeres during G1, a considerable but transient increase was detected during early mitosis. A low level of Cid loading was detected in late S and G2, starting at the reported time of centromere DNA replication. Our results reveal the complexities of Drosophila centromere protein dynamics and its intricate coordination with cell cycle progression.

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Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

Cited by 185 publications

References 20 publications

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells

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