Characterisation and high-resolution distribution of a matrix attachment region-binding protein (MFP1) in proliferating cells of onion

The first matrix attachment region (MAR)-binding protein sequenced in plants, MFP1, has been characterised in two dicot species. Based on their antigenic relationship, we report here the conservation of MFP1-like proteins in proliferating root cells of onion (Allium cepa L). Two MFP1-like proteins with different molecular masses and solubilities were detected. The most abundant was a 90-kDa basic protein, presenting several separate spots in two-dimensional blots. The MFP1 was partially soluble and, similar to the proliferating cell nuclear antigen (PCNA)-labelled replication factories in the nucleus and nuclear matrix, was localised at discrete foci as detected by confocal microscopy. High-resolution immunolocalisation of MFP1 by electron microscopy identified the foci as nuclear structures, some of them containing PCNA, which are ultrastructurally similar to the replication factories described in animal cells. Our data provide the first report on MFP1-like proteins in the Alliaceae. In addition, we present evidence of the presence of AcMFP1 in the putative replication factories. Received: 12 May 2000 / Accepted: 13 September 2000     

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.     

CK2 phosphorylation weakens 90 kDa MFP1 association to the nuclear matrix in Allium cepa

MFP1 is a conserved plant coiled-coil protein located on the stroma side of the chloroplast thylakoids, as well as in the nuclear matrix. It displays species-specific variability in the number of genes, proteins, and expression. Allium cepa has two nuclear proteins antigenically related to MFP1 with different M(r), pI, distribution, and expression, but only the 90 kDa MFP1 protein is a nuclear matrix component that associates with both the nucleoskeletal filaments and a new category of nuclear bodies. The 90 kDa AcMFP1 migrates in two-dimensional blots as two sets of spots. The hypo-phosphorylated forms (pI approximately 9.5) are tightly bound to the nuclear matrix, while high ionic strength buffers release the more acidic hyper-phosphorylated ones (pI approximately 8.5), suggesting that the protein is post-translationally modified, and that these modifications control its attachment to the nuclear matrix. Dephosphorylation by exogenous alkaline phosphatase and phosphorylation by exogenous CK2, as well as specific inhibition and stimulation of endogenous CK2 with heparin and spermine and spermidine, respectively, revealed that the protein is an in vitro and in vivo substrate of this enzyme, and that CK2 phosphorylation weakens the strength of its binding to the nuclear matrix. In synchronized cells, the nuclear 90 kDa AcMFP1 phosphorylation levels vary during the cell cycle with a moderate peak in G2. These results provide the first evidence for AcMFP1 in vivo phosphorylation, and open up further research on its nuclear functions.     

MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

Dynamics and interactions of parvoviral NS1 protein in the nucleus

Nuclear positioning and dynamic interactions of viral proteins with nuclear substructures play essential roles during infection with DNA viruses. Visualization of the intranuclear interactions and motility of the parvovirus replication protein (NS1) in living cells gives insight into specific parvovirus protein-cellular structure interactions. Confocal analysis of highly synchronized infected Norden Laboratory Feline Kidney cells showed accumulation of nuclear NS1 in discrete interchromosomal foci. NS1 fused with enhanced yellow fluorescence protein (NS1-EYFP) provided a marker in live cells for dynamics of NS1 traced by photobleaching techniques. Fluorescence Recovery after Photobleaching suggested that the NS1 protein is not freely diffusing but undergoes transient interactions with nuclear compartments. Fluorescence Loss in Photobleaching demonstrated for the first time the shuttling of a parvoviral protein between the nucleus and the cytoplasm as assayed with NS1-EYFP. Finally, time-lapse imaging of infected cells revealed that the intranuclear distribution of NS1-EYFP evolves dramatically starting from the formation of NS1 foci and proceeding to a homogenous distribution extending throughout the nucleus.     

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.     

Integrin signalling regulates the nuclear localization and function of the lysophosphatidic acid receptor-1 (LPA1) in mammalian cells

We show that LPA1 (lysophosphatidic acid receptor-1) is constitutively localized in the nucleus of mammalian cells. LPA1 also traffics from cell membranes to the nucleus in response to LPA (lysophosphatidic acid). Several lines of evidence suggest an important role for cell-matrix interaction in regulating the constitutive nuclear localization of LPA1. First, the RGDS peptide, which blocks cell matrix-induced integrin clustering and cytoskeletal rearrangement, reduced the number of cells containing LPA1 in the nucleus. Secondly, a higher proportion of cells contained nuclear LPA1 when adhesion on fibronectin-coated glass was compared with adherence to polylysine-coated glass. Thirdly, pre-treatment of cells with the Rho kinase inhibitor (Y27632) or the myosin light chain kinase inhibitor (ML9) reduced the number of cells containing nuclear LPA1. The addition of LPA and/or Ki16425 (which binds to LPA1) to isolated nuclei containing LPA1 induced the phosphorylation of several proteins with molecular masses of 34, 32, 14 and 11 kDa. These findings demonstrate that trafficking of LPA1 to the nucleus is influenced by cell-matrix interactions and that nuclear LPA1 may be involved in regulating intranuclear protein phosphorylation and signalling.     

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.     

Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo

The human immunodeficiency virus type 1 (HIV-1) protein Vpr (viral protein R) arrests cells in the G2 phase of the cell cycle, a process that requires activation of the ATR (ataxia-telangiectasia and Rad3-related) pathway. In this study we demonstrate that the expression of Vpr does not cause DNA double-strand breaks but rather induces ATR activation, as indicated by induction of Chk1 phosphorylation and the formation of gamma-H2AX and 53BP1 nuclear foci. We define a C-terminal domain containing repeated H(F/S)RIG sequences required for Vpr-induced activation of ATR. Further investigation of the mechanism by which Vpr activates the ATR pathway reveals an increase in chromatin binding of replication protein A (RPA) upon Vpr expression. Immunostaining shows that RPA localizes to nuclear foci in Vpr-expressing cells. Furthermore, we demonstrate direct binding of Vpr to chromatin in vivo, whereas Vpr C-terminal domain mutants lose this chromatin-binding activity. These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication.     

Iron containing nuclear inclusions in human pigmentary cirrhosis (author's transl)]

In human pigmentary cirrhosis nuclear (pseudo-)inclusions of cytoplasmic material, containing less or more degenerated and therefore faintly stained hemosiderin granules, are to be observed. But sometimes there are also finely fibrillar or granular proteinaceous materials, stainable by the Prussian-blue reaction, lying between the chromatin-strands or occupying the whole nucleus and displacing the chromatin to the nuclear envelope (margination of chromatin). Such uncoloured substances may condense into homogeneous masses and nearly hexagonal (0r related) crystals with a diameter up to 14 micron and a yellow-brownish colour, giving a strongly positive PERL's reaction. In contrast to the preceding stages intranuclear crystals of this kind have been observed in one case only. After destruction of the nuclear envelope and the marginated chromatin the crystals are lying free in the cytoplasm and later on, the cytoplasm being destroyed too, they may be ingested by von Kupffer cells. All the iron containing crystals, to be found in the cytoplasm, derive from former intranuclear inclusions. The intranuclear deposits of iron containing protein are interpreted as ferritin-aggregates. It is supposed that ferritin molecules, built up in the cytoplasm, do enter the nucleus via the pores of the nuclear envelope. Such an event not only signalizes a cytopathologic reaction but in turn may give rise to such additional cytopathologic lesions as cell shrinking and cell death.     

Mapping replicational sites in the eucaryotic cell nucleus

We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus.     

AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region

Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones.     

ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation

Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.