Acceptor-photobleaching FRET analysis of core kinetochore and NAC proteins in living human cells

Hellwig, Daniela; Hoischen, Christian; Ulbricht, Tobias; Diekmann, Stephan

doi:10.1007/s00249-009-0498-x

Cited by 12 publications

(16 citation statements)

References 43 publications

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“…These data imply that the N-terminus of CENP-N is in close proximity to the N-terminus of CENP-A. By contrast, we detected no FRET between EGFP-CENP-N and mCherry-CENP-C (Hellwig et al, 2009) and between EYFP-CENP-C and Cerulean-CENP-A (S. Orthaus, Towards the architecture of the human kinetochore, PhD thesis, Friedrich Schiller University, Jena, Germany, 2006). These results are consistent with recent findings on the kinetochore binding of CENP-C (Przewloka et al, 2011;Screpanti et al, 2011).…”

Section: Cenp-n Binds In Close Proximity To Cenp-a In Vivomentioning

confidence: 76%

“…During bleaching of spot 2, the donor fluorescence intensity significantly increased (Fig. 1B, black arrow) indicating that FRET occurs between EGFP-CENP-N and mCherry-CENP-A (Hellwig et al, 2009). Careful quantification indicated that such FRET transfer occurred in 64% of the cases (P,0.001; Fig.…”

Section: Cenp-n Binds In Close Proximity To Cenp-a In Vivomentioning

confidence: 96%

“…However, whether CENP-A and CENP-N are closely associated in vivo is not known. To analyse the binding of cellular CENP-N to the CENP-Acontaining nucleosome in centromeric chromatin, we performed a FRET-based approach, which we have previously utilised to study protein proximities and interactions in cells (Hellwig et al, 2009;Orthaus et al, 2008;Orthaus et al, 2009). We expressed a full-length human CENP-N fusion with EGFP and confirmed that the EGFP-CENP-N colocalised to kinetochores with CENP-A in human HEp-2 cells (Fig.…”

Section: Cenp-n Binds In Close Proximity To Cenp-a In Vivomentioning

confidence: 99%

“…Into the resulting purified 4.433 bp fragment, we ligated a 713 bp AgeI-BsrGI fragment resulting from the vector pmCherry-C1 (Orthaus et al, 2009). For expressing human CENP-N fused to the C-terminus of EGFP, we used vector pH-G-CENP-N (Hellwig et al, 2009). All clones were verified by sequencing (MWG Biotech, Ebersberg, Germany).…”

Section: Plasmidsmentioning

confidence: 99%

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Dynamics of CENP-N kinetochore binding during the cell cycle

Hellwig¹,

Emmerth

Ulbricht³

et al. 2011

Journal of Cell Science

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SummaryAccurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

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Section: Cenp-n Binds In Close Proximity To Cenp-a In Vivomentioning

confidence: 76%

Section: Cenp-n Binds In Close Proximity To Cenp-a In Vivomentioning

confidence: 96%

Section: Cenp-n Binds In Close Proximity To Cenp-a In Vivomentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

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Dynamics of CENP-N kinetochore binding during the cell cycle

Hellwig¹,

Emmerth

Ulbricht³

et al. 2011

Journal of Cell Science

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“…FRET measurements were performed as previously described with some modifications (Hellwig et al 2009;Bui et al 2012). Cells were labeled with 0.6 µM TMR-Star and 3 µM Oregon Green using the methods described above.…”

Section: Snap-labeling Ab-fret Measurementsmentioning

confidence: 99%

Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N

et al. 2015

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Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.

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