Renato Bareggi

The nuclear matrix and apoptosis.

Abstract Apoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells.

Rab3A and Rab3D Control the Total Granule Number and the Fraction of Granules Docked at the Plasma Membrane in PC12 Cells

Rab proteins are Ras‐like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno‐electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild‐type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

Abstract: Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation.

Molecular characterization of protein kinase C-α binding to lamin A

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)‐binding protein. Here, we have identified the regions of PKC‐α that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione‐S‐transferase (GST) fused to elements of rat PKC‐α, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium‐dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200–217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC‐α to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca2+. We have also found that the binding site of lamin A for the CaLB domain of PKC‐α is localized in the carboxyl‐terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme‐specific drugs to attenuate or reverse PKC‐dependent nuclear signaling pathways important for the pathogenesis of cancer.

Re-examination of the mechanisms regulating nuclear inositol lipid metabolism

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3‐kinase and phosphoinositide‐specific phospholipase C‐β1.

Insulin-like growth factor-I-dependent stimulation of nuclear phospholipase C-?1 activity in Swiss 3T3 cells requires an intact cytoskeleton and is paralleled by increased phosphorylation of the phospholipase

Swiss 3T3 mouse fibroblasts were exposed to 10 μM colchicine to disrupt microtubules, then stimulated with insulin‐like growth factor‐I. Immunoprecipitation experiments showed that insulin‐like growth factor‐I receptor and insulin receptor substrate‐1 were tyrosine phosphorylated to the same extent in both cells treated with colchicine and in those not exposed to the drug. Moreover, the activity of phosphatidylinositol 3‐kinase was not affected by incubation with colchicine. While in nuclei prepared from cells not exposed to colchicine it was possible to detect an insulin‐like growth factor‐I‐dependent increase in the mass of diacylglycerol, as well as stimulation of phospholipase C activity, no similar changes were observed in nuclei obtained from cells treated with colchicine. Activation of the nuclear phospholipase activity was paralleled by an increase of its phosphorylation. Immunofluorescent studies revealed that mitogen‐activated protein kinase did not translocate towards the nucleus when the cytoskeleton was depolymerized. These results show that in Swiss 3T3 cells some as yet unknown events necessary for the insulin‐like growth factor‐I‐dependent activation of nuclear polyphosphoinositide metabolism require the presence of an intact cytoskeleton and are situated down‐stream the activation of insulin receptor substrate‐1 and phosphatidylinositol 3‐kinase. Activation of nuclear phospholipase C‐β1 might be linked to its phosphorylation and translocation of mitogen‐activated protein kinase to the nucleus. J. Cell. Biochem. 72:339–348, 1999.

Nuclear changes in necrotic HL-60 cells

Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptosis is characterized by well‐defined nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On the other hand, the nuclear modifications that occur during necrosis are largely less known. Here, we have investigated whether or not nuclear modifications occur during ethanol‐induced necrotic cell death of HL‐60 cells. By means of immunofluorescence staining, we demonstrate that the patterns given by antibodies directed against some nuclear proteins (lamin B1, NuMA, topoisomerase IIα, SC‐35, B23/nucleophosmin) changed in necrotic cells. The changes in the spatial distribution of NuMA strongly resembled those described to occur during apoptosis. On the contrary, the fluorescent pattern characteristic for other nuclear proteins (C23/nucleolin, UBF, fibrillarin, RNA polymerase I) did not change during necrosis. By immunoblotting analysis, we observed that some nuclear proteins (SAF‐A, SATB1, NuMA) were cleaved during necrosis, and in the case of SATB1, the apoptotic signature fragment of 70 kDa was also present to the same extent in necrotic samples. Caspase inhibitors did not prevent proteolytic cleavage of the aforementioned polypeptides during necrosis, while they were effective if apoptosis was induced. In contrast, lamin B1 and topoisomerase IIα were uncleaved in necrotic cells, whereas they were proteolyzed during apoptosis. Transmission electron microscopy analysis revealed that slight morphological changes were present in the nuclear matrix fraction prepared from necrotic cells. However, these modifications (mainly consisting of a rarefaction of the inner fibrogranular network) were not as striking as those we have previously described in apoptotic HL‐60 cells. Taken together, our results indicate that during necrosis marked biochemical and morphological changes do occur at the nuclear level. These alterations are quite distinct from those known to take place during apoptosis. Our results identify additional biochemical and morphological criteria that could be used to discriminate between the two types of cell death. J. Cell. Biochem. Suppl. 36: 19–31, 2001.

Behavior of nucleolar proteins during the course of apoptosis in camptothecin‐treated HL60 cells

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin‐treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti‐nucleolar antibodies seen in various pathological disorders. J. Cell. Biochem. 78:264–277, 2000.

A quantitative study on the spatial and temporal ossification patterns of vertebral centra and neural arches and their relationship to the fetal age

A double-staining technique on 37 human embryos and fetuses (crown-rump length, CRL, between 38 and 116 mm) has been performed to study the ossification patterns of the vertebral column. Different growth sequences for centra and neural arches were observed. The survey of ossified centers suggested it was possible to relate significantly their appearance with the CRL. On the basis of already known data defining the developmental age in relationship to the latter parameter, we suggest their numerical evaluation as a further parameter for the assessment of the fetal age. Therefore, we have worked out a table that may be used either to determine the normal fetal growth, or when other parameters cannot be relied upon (i.e. in morphological diseases) for this aim.

Diffuse vesicular distribution of Rab3D in the polarized neuroendocrine cell line AtT-20

The neuroendocrine cell line AtT‐20 has two types of storage vesicles: dense core granules and synaptic vesicles, both sequestered at the tip of the processes. Here we show that Rab3D protein, which is abundant in fat cells, is also expressed in AtT‐20 cells. Differently from Rab3A, which is localized in secretory vesicles accumulated at the tips, Rab3D has a diffuse vesicular distribution in the cytoplasm of the cell body, the processes and the tips. In AtT‐20 cells, Rab3D may define a regulated secretory pathway which functions independently from cell polarity.