Loss of lamin A function increases chromatin dynamics in the nuclear interior.

Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.

Transient anomalous diffusion of telomeres in the nucleus of mammalian cells.

We measured individual trajectories of fluorescently labeled telomeres in the nucleus of eukaryotic cells in the time range of 10(-2)-10(4)sec by combining a few acquisition methods. At short times the motion is subdiffusive with r2 approximately talpha and it changes to normal diffusion at longer times. The short times diffusion may be explained by the reptation model and the transient diffusion is consistent with a model of telomeres that are subject to a local binding mechanism with a wide but finite distribution of waiting times. These findings have important biological implications with respect to the genome organization in the nucleus.

Nuclear relocalization of the pre-mRNA splicing factor PSF during apoptosis involves hyperphosphorylation, masking of antigenic epitopes, and changes in protein interactions.

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1--70K and SR proteins and therefore may acquire new functions.

Reorganization of nuclear factors during myeloid differentiation.

Differentiation in several stem cell systems is associated with major morphological changes in global nuclear shape. We studied the fate of inner-nuclear structures, splicing factor-rich foci and Cajal (coiled) bodies in differentiating hemopoietic, testis and skin tissues. Using antibodies to the splicing factors PSF, U2AF(65) and snRNPs we find that these proteins localize in foci throughout the nuclei of immature bone marrow cells. Yet, when granulocytic cells differentiate and their nuclei condense and become segmented, the staining localizes in a unique compact and thread-like structure. The splicing factor-rich foci concentrate in the interior of these nuclei while the nuclear periphery and areas of highly compact chromatin remain devoid of these molecules. Differentiated myeloid cells do not stain for p80 coilin, the marker for Cajal bodies. Immature myeloid cells contain Cajal bodies although these usually do not coloclaize with PSF-rich foci. Following complete inhibition of transcription in myeloid cells, the threaded PSF pattern becomes localized in several foci in the different lobes of mature granulocytes while in human HL-60 immature myeloid leukemia cells PSF is found in the perinucleolar compartment. Studies of other differentiating stem cell systems show that PSF staining disappears completely in differentiated, transcriptionally inactive sperm cells, is scarce as cells migrate from the inner skin layers outward and is lost as cells of the hair follicle mature. We conclude that the formation and distribution of splicing factor-rich foci in the nucleus during differentiation of various cell lineages is dependent on the levels of chromatin condensation and the differentiation status of the cell.

Enhanced proteolysis of pre-mRNA splicing factors in myeloid cells.

OBJECTIVE: Molecular identification and characterization of the bone marrow nuclear protein detected by the B92 monoclonal antibody. MATERIALS AND METHODS: The protein was purified to homogeneity from acute myeloid leukemia cells and was subjected to peptide digestion and amino acid sequencing. Identified sequences were used to screen a bone marrow cDNA library in search of matching transcripts. The protein was further studied in different cells and tissues by examination of protease inhibitors and harsh lytic conditions and during apoptosis in HL-60 cells. RESULTS: We found that the apparent bone marrow specific protein is a 47 kD proteolytic cleavage product of PSF, an essential pre-mRNA splicing factor. PSF is completely cleaved to p47 during lysis of immature myeloid cells due to potent proteolytic activity found in these cells but is rare in other cells and tissues. Furthermore, p47 is abundant in intact normal and tumor myeloid cells while in other cell types it is undetectable. The cleavage of PSF is accompanied by digestion of the PTB splicing regulator but not other proteins tested. In contrast, during apoptosis PTB is degraded while PSF remains intact. CONCLUSIONS: The bone marrow 47 kD protein is a fragment constituting the N-terminal, protease-resistant half of the splicing factor PSF. Proteolytic degradation of PSF specifically occurs in intact myeloid cells and this process is enhanced upon myeloid cell lysis.

Adhesion molecules involved in the interactions between early T cells and mesenchymal bone marrow stromal cells.

We previously reported that among the various thymic lymphocyte subpopulations, the immature T cells preferentially adhere to mesenchymal bone marrow stroma. In the present study we examined the interactions between phenotypically defined populations of early T cells and stromal cell lines. The immature T cells segregated into two subpopulations according to their adhesive capacity. Whereas the majority of the adherent CD4-CD8- T cells were devoid of CD3/TCRalphabeta, most of the nonadherent CD4-CD8- T cells expressed this receptor complex. The adhesion of T cells to bone marrow stroma almost entirely was accounted for by CD49d and CD90, whereas that of adherent CD4-CD8- cells also was dependent on CD44, CD62L, and CD117 receptor. Blocking antibody combinations failed to reduce the adherence of these early T cells to less than 50% that of the control. On the other hand, the adhesion of unselected thymocytes to the stroma was reduced by 80%, using the same blocking antibodies. Therefore, the participation of additional molecules in the adhesion of early T cells to mesenchymal stroma is implicated. Comparison between the interaction of T cells with bone marrow mesenchymal or with thymus-derived epithelial stroma indicated that T cells utilize a selected set of adhesion molecules under each situation. Although CD49d and CD90 participated in both cases, CD11a, CD18, and CD2 receptors played a dominant role in the adhesion of T cells to thymic epithelium only. This study may point to a role of mesenchymal stroma in the regulation of early T-cell lymphopoiesis in the bone marrow.

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

Nuclear antigen expressed by proliferating cells.

We describe a novel mouse monoclonal antibody (PRA-72) that recognizes a nuclear antigen associated with cell proliferation. The monoclonal antibody stained the nuclei of logarithmically growing cultured stromal cells. The nuclear staining disappeared when these cells entered Gzero phase of the cell cycle. Western blot analysis revealed a nuclear protein which appeared as a doublet at 35-40 KD, which was undetectable in extracts from confluent cells. Immunocytological study of purified cell populations from various cell cycle phases revealed peripheral nuclear staining in all stages except mitosis, when the chromosomes were observed enveloped with the antigen. In co-cultures of quiescent stromal cells and proliferating hemopoietic precursors, only the latter showed nuclear staining by PRA-72 monoclonal antibody. Further indications for selective expression of the antigen by proliferating cells were found by an immunohistochemical study of various tissues including newborn mouse bone marrow and its surrounding connective tissue, mouse tongue epithelium, and human carcinoma of the colon. This antibody may, therefore, prove useful in the evaluation of human tumors.

A hematopoietic organ-specific 49-kD nuclear antigen: predominance in immature normal and tumor granulocytes and detection in hematopoietic precursor cells.

A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a novel mouse monoclonal antibody (B92) raised against stromal cells. The protein was found in the immunizing cells using a sensitive method. However, its detection in the bone marrow by the B92 antibody seemed to stem from the abundance of p49 in immature cells of the myeloid lineage. Study of the bone marrow following in vivo irradiation or 5-fluorouracil (5-FU) treatment, in vitro culture with differentiation-inducing factors and long-term culture, and cell sorting all pointed in the same direction: the protein was found in early myeloid cells and in hematopoietic precursor cells. These results were in accordance with the specific presence of p49 in primary radiation-induced myeloid leukemia and its absence in spontaneous B lymphoma. Immunofluorescent staining using B92 antibody detected a nuclear antigen forming a dotted pattern in early myeloid cells and day 12 colony-forming units-spleen (CFU-S). Nuclear localization of p49 was further demonstrated by subcellular fractionation followed by Western blotting. We thus identified a nuclear protein that within the hematopoietic population is detected in hematopoietic precursor cells, predominates in early myeloid cells, and is reduced following differentiation. These properties imply that p49 might be involved in the regulation of hematopoietic cell growth or differentiation.

The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. Identification as a stroma-derived activin A.

A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokine-deprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.