In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

The Hutchinson–Gilford progeria syndrome (HGPS) is a premature aging disease caused by mutations of the LMNA gene leading to increased production of a partially processed form of the nuclear fibrillar protein lamin A – progerin. Progerin acts as a dominant factor that leads to multiple morphological anomalies of cell nuclei and disturbances in heterochromatin organization, mitosis, DNA replication and repair, and gene transcription. Progerin-positive cells are present in primary fibroblast cultures obtained from the skin of normal donors at advanced ages. These cells display HGPS-like defects in nuclear morphology, decreased H3K9me3 and HP1, and increased histone H2AX phosphorylation marks of the DNA damage loci. Inhibition of progerin production in cells of aged non-HGPS donors in vivo increases the proliferative activity, H3K9me3, and HP1, and decreases the senescence markers p21, IGFBP3, and GADD45B to the levels of young donor cells. Thus, progerin-dependent mechanisms act in natural aging. Excessive activity of the same mechanisms may well be the cause of premature aging in HGPS. Telomere attrition is widely regarded to be one of the primary hallmarks of aging. Progerin expression in normal human fibroblasts accelerates the loss of telomeres. Changes in lamina organization may directly affect telomere attrition resulting in accelerated replicative senescence and progeroid phenotypes. The chronological aging in normal individuals and the premature aging in HGPS patients are mediated by similar changes in the activity of signaling pathways, including downregulation of DNA repair and chromatin organization, and upregulation of ERK, mTOR, GH-IGF1, MAPK, TGFβ, and mitochondrial dysfunction. Multiple epigenetic changes are common to premature aging in HGPS and natural aging. Recent studies showed that epigenetic systems could play an active role as drivers of both forms of aging. It may be suggested that these systems translate the effects of various internal and external factors into universal molecular hallmarks, largely common between natural and accelerated forms of aging. Drugs acting at both natural aging and HGPS are likely to exist. For example, vitamin D3 reduces the progerin production and alleviates most HGPS features, and also slows down epigenetic aging in overweight and obese non-HGPS individuals with suboptimal vitamin D status.

The mechanisms by which the supramolecular order is formed inside the cell nucleus remain poorly understood. So far, two major hypotheses - ordered assembly and stochastic self-organization - have been discussed. To determine which mechanism is responsible for the formation of nuclear envelope, cells overexpressing one of the nuclear envelope proteins (lamin A, lamin B1, pom121 or ndc1) were investigated. According to the ordered assembly model, the presence of an excessive amount of a component has no effect in the formation of the normal structure of a nuclear envelope because it is programmed and cannot be distorted. In contrast, according to the self-organization concept, there is no such strictly determined cellular structures, and an excessive amount of even one component will affect the cellular organization. In the present study, formation of a redundant nuclear envelope was observed in the case of lamin B1 and lamin A overexpression. In the case of the nucleoporins pom121 and ndc1, no incorporation of the overexpressed proteins into the nuclear envelope was observed on the first day after transfection; however, the remodeling of endoplasmic reticulum elements and the formation of membrane aggregates in the cytoplasm were observed. After mitosis, pom121 from the cytoplasmic aggregates was translocated into the redundant nuclear envelope in which it induced inner nuclear membrane protrusions. Therefore, our results indicate that the formation of the nuclear envelope is not predetermined and that an excessive amount of even one protein component can affect cellular structure formation. This study concluded that nuclear envelope formation is achieved by the self-organization mechanism.

In vitro radioligand assay revealed interaction of afobazole with sigma(1)-receptors (Ki=5.9x10(-6) M). Translocation of sigma(1)-receptors from the endoplasmic reticulum to the outer membrane was demonstrated by confocal microscopy. Experiments were performed on the model of HT-22 immortalized hippocampal cells after incubation with afobazole in a concentration of 10(-8) M.

The nuclear lamina represents a multifunctional platform involved in such diverse yet interconnected processes as spatial organization of the genome, maintenance of mechanical stability of the nucleus, regulation of transcription and replication. Most of lamina activities are exerted through tethering of lamina-associated chromatin domains (LADs) to the nuclear periphery. Yet, the lamina is a dynamic structure demonstrating considerable expansion during the cell cycle to accommodate increased number of LADs formed during DNA replication. We analyzed dynamics of nuclear growth during interphase and changes in lamina structure as a function of cell cycle progression. The nuclear lamina demonstrates steady growth from G1 till G2, while quantitative analysis of lamina meshwork by super-resolution microscopy revealed that microdomain organization of the lamina is maintained, with lamin A and lamin B microdomain periodicity and interdomain gap sizes unchanged. FRAP analysis, in contrast, demonstrated differences in lamin A and B1 exchange rates; the latter showing higher recovery rate in S-phase cells. In order to further analyze the mechanism of lamina growth in interphase, we generated a lamina-free nuclear envelope in living interphase cells by reversible hypotonic shock. The nuclear envelope in nuclear buds formed after such a treatment initially lacked lamins, and analysis of lamina formation revealed striking difference in lamin A and B1 assembly: lamin A reassembled within 30 min post-treatment, whereas lamin B1 did not incorporate into the newly formed lamina at all. We suggest that in somatic cells lamin B1 meshwork growth is coordinated with replication of LADs, and lamin A meshwork assembly seems to be chromatin-independent process.