Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin

Abstract

BackgroundProtein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D.ResultsWe show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci.ConclusionBased on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.

Transcription regulates telomere dynamics in human cancer cells

“…Mammalian telomeres are not immobile, but, as other chromatin loci, move within the nuclear territory (Molenaar et al 2003;Dimitrova et al 2008;Wang et al 2008;Jegou et al 2009;Stixova et al 2011). The motility of individual telomeres within the same cell is largely heterogeneous and inversely correlates with their length.…”

Micron-scale coherence in interphase chromatin dynamics

“…To date, studies of chromatin dynamics consist largely of photobleaching (13)(14)(15)(16) and photoactivation techniques (17,18), which visualize turnover of nuclear proteins and movement of whole sections of the nucleus, and tracking of chromatin-attached single particles to quantify movement dynamics (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Single particles within chromatin that have been tracked include fluorescently labeled single-DNA sites (19)(20)(21)(22), nuclear proteins (23)(24)(25)(26), chromosome territories (27,28) and subchromosomal foci (29,30).…”

“…Single particles within chromatin that have been tracked include fluorescently labeled single-DNA sites (19)(20)(21)(22), nuclear proteins (23)(24)(25)(26), chromosome territories (27,28) and subchromosomal foci (29,30). These studies revealed dynamics that can be largely described as diffusive or subdiffusive with constraint, with few instances showing an apparent directional movement for single genes (19,20).…”

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

“…We applied methods similar to other studies that revealed the movement of nuclear bodies or protein foci. 17,27,29,30 We used contour-based image registration techniques 31 and specifically tailored tracking and segmentation algorithms (Figs. 2A-D, 3D and 4A, B).…”

“…The following methods can be applied for such analyses: 1] FRAP analysis of protein diffusion at DNA lesions, 2] advanced image processing algorithms to follow DNA repair foci trajectories, and 3] image analysis to describe morphology of protein-abundant DNA lesions. [15][16][17][18] Critical parameters also include the particle dynamics and fluorescence signal intensity of radiation-damaged chromatin, which can be monitored in 3 dimensions over time. Thus, the use of above mentioned experimental approaches can allow for the proper characterization of DNA lesion behavior in space and time.…”