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Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.

Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.

Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme d-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo. [ABSTRACT FROM AUTHOR]

The ability to determine the time since forming of seized nuclear material would provide crucial data to be used in its investigation. The time dependent processes of diffusion relevant to this need are examined; to include grain boundary diffusion, and discontinuous precipitation, and an assessment is performed of the utility of examination of impurity and alloying element concentration profiles for use as a pre-detonation nuclear forensics tool for determining the age of a metal sample. Several examples illustrate the effects of time on both binary alloys and multi-component systems. Parallels are drawn from studies of diffusion in mineral samples under geologic time and proposes modifications to that approach in order to determine the time since a metal sample was cast or formed.

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance. [ABSTRACT FROM AUTHOR]

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility. [ABSTRACT FROM AUTHOR]

Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression., Competing Interests: FP, ZL, PD, CH, ES, AR, NH No competing interests declared, RT One of the three founding funders of eLife and a member of eLife's Board of Directors, (© 2019, Piccolo et al.)