Mechanisms of nuclear lamina growth in interphase.

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.

Poly(ADP-ribosyl)ation of mannose-binding lectin out of human kidney cells.

Mannose-binding lectin was identified as a substrate of tankyrase 2, an enzyme that catalyzes poly(ADP-ribosyl)ation. The endogenous tankyrase 2 was isolated out of cytoplasm of human embryonic kidney cells. It was bound to a soluble complex of at least two other proteins; they were identified using specific antibodies and other approaches as keratin 1 and mannose-binding lectin. Using immunoblot analysis and radioactive labeling, we detected tankyrase-2-dependent poly(ADP-ribosyl)ation of mannose-binding lectin. In the presence of NAD(+), the complex of keratin 1 and lectin was dissociated, what was recorded during elution of its separate components out of affinity columns and by decrease of their apparent molecular masses during gel-filtration. Tankyrase 2 also inhibited the carbohydrate-binding function of the lectin. The latter effect was observed using mannose-binding lectin out of human serum, which is free from keratin 1. As a result of tankyrase-2 activity, the lectin lost its affinity to mannan-agarose. The discovery of this new biochemical mechanism justifies further analysis of its physiological and medical significance.

Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells.

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.