Anthony D. Mills

Nuclear protein migration involves two steps: Rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.

Distinct functions for the two importin subunits in nuclear protein import

THE import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs)1,2, energy3,4 and soluble factors5, namely importin-α(Mr 60K)6-12,28, importin-β (90K)8-11,13 and Ran14,15. Importin-α is primarily responsible for NLS recognition6-12,29 and is a member of a protein family that includes the essential yeast nuclear pore protein SRPlp (ref. 16). As the first event, the complex of importin-α and importin-β binds the import substrate in the cytosol8,9. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-β. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-α in the nucleus. In contrast, importin-β accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-β on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

Cdc6 protein causes premature entry into S phase in a mammalian cell-free system.

We exploit an improved mammalian cell‐free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

mRNA Export from Mammalian Cell Nuclei Is Dependent on GANP

Summary Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY [1, 2] and SR splicing factors [3] and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins [4–10]. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)+RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.

Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis.

Plasmid R1 inhibits growth of bacteria by synthesizing an inhibitor of cell proliferation, Kid, and a neutralizing antidote, Kis, which binds tightly to the toxin. Here we report that this toxin and antidote, which have evolved to function in bacteria, also function efficiently in a wide range of eukaryotes. Kid inhibits cell proliferation in yeast, Xenopus laevis and human cells, whilst Kis protects. Moreover, we show that Kid triggers apoptosis in human cells. These effects can be regulated in vivo by modulating the relative amounts of antidote and toxin using inducible eukaryotic promoters for independent transcriptional control of their genes. These findings allow highly regulatable, selective killing of eukaryotic cells, and could be applied to eliminate cancer cells or specific cell lineages in development.

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes

We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoplasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60%), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.

Detection of S-phase cells in tissue sections by in situ DNA replication

ere we demonstrate a rapid and simple method for identifying Sphase cells in tissue sections or cell cultures without the need to prelabel them with radioactive or halogenated precursors before sample collection. When sections of snap-frozen tissue samples or frozen, permeabilized cell cultures grown on coverslips are incubated in a buffer containing ribonucleoside triphosphates and deoxyribonucleoside triphosphates, S-phase cells can be identified because their nuclei continue to synthesize DNA in situ. Incubations as short as 15 min allow the detection of S-phase cells by fluorescent microscopy. This method has many potential applications in cell and developmental biology and in medicine, especially cancer diagnosis. While developing cell-free DNA-replication systems from human cells, we observed that frozen nuclei from S-phase cells continue to synthesize DNA in vitro after they are rethawed and incubated in buffers containing precursors. The method described here exploits this observation to detect S-phase cells in frozen tissue sections by virtue of their DNA replication in situ. To test this potential assay for S-phase cells, we permeabilized HeLa cells grown on coverslips and overlaid the cells with an incubation buffer containing ribonucleoside triphosphates (NTPs), deoxyribonucleoside triphosphates (dNTPs) and bovine serum albumin (BSA) (Fig. 1). DNA synthesis was monitored by the incorporation of a labelled nucleotide precursor and with the use of fluorescent confocal microscopy (Fig. 1). Figure 1 shows that HeLa cells in S phase, but not other phases, continue to synthesize DNA both in situ and in vitro. S-phase cells were prelabelled in vivo with bromodeoxyuridine (BrdU, a marker of DNA synthesis; red), permeabilized and labelled in vitro for 1 h at 37 °C using Digoxygenin-11-dUTP (Dig–dUTP, green). DNA was stained using TOTO-3 and appears blue in the three-channel merged image (Fig. 1d). Figure 1c is a merged image of Fig. 1a, b and shows that those nuclei that were replicating their DNA in vivo before permeabilization (as revealed by BrdU staining, Fig. 1a) were the same as those that were labelled in vitro (with Dig–dUTP, Fig. 1b), whereas cells that were unlabelled in vivo remained unlabelled in vitro (blue nuclei, Fig. 1d). At a higher magnification, newly replicated DNA showed the punctate pattern typical of incorporation into replication foci. Individual foci replicating in vitro (Fig. 1f) coincided with those that incorporated BrdU in vivo (Fig. 1e), giving yellow foci in the merged image (Fig. 1g, h). The observation that no purely green replication foci were present in the merged image (Fig. 1g, h) supports the idea that only DNA elongation is occurring, and not the initiation of DNA replication. When S-phase HeLa cells were ‘mock-permeabilized’ by omitting digitonin from the phosphate-buffered saline (see Methods), they did not incorporate Dig–dUTP detectably in vitro (data not shown). To confirm that only permeabilized S-phase cells incorporated labelled dNTPs in vitro, we studied permeabilized quiescent Wi38 cells. Less than 2% of these cells were labelled in vitro; this percentage is the same as the background percentage of S-phase cells seen by BrdU incorporation in vivo (data not shown). Figure 1 shows that S-phase cells can be selectively detected by virtue of their DNA replication in situ; quiescent (G0) or other non-S-phase cells were not labelled. Furthermore, the intranuclear sites that are used for DNA replication in vitro are the same as those that are active in vivo (Fig. 1e–g). We then examined frozen tissue sections. Sections were quickthawed, overlaid with the same incubation buffer as that used for permeabilized tissue-culture cells, and incubated as before. As the sections were of subcellular thickness, the permeabilization step was unnecessary. After incubation, sections were stained either for fluorescent microscopy, using propidium iodide to label DNA (red) and fluorescein-tagged anti-digoxygenin antibody to detect incorporated Dig–dUTP (green; appears yellow in the merged image) (Fig. 2b–h), or for bright-field microscopy, using anti-digoxygenin antibody linked to horseradish peroxidase (HRP, brown) and haematoxylin counterstain (Fig. 2i). The whole process for frozen tissue sections is summarized in Fig. 2a. Figure 2 also shows that this simple method easily identifies S-phase cells. Incubation times as short as 15 min were sufficient for the labelled DNA precursor to be detected in some nuclei. Figure 2b shows sections of normal ectocervix with only a small minority of labelled cells in the basal or parabasal layers. Figure 2c shows a premalignant lesion, cervical intraepithelial neoplasia grade 3 (equivalent to high-grade squamous intraepithelial lesion), with cells undergoing DNA replication throughout the thickness of the tissue. These results partly resemble data that we obtained previously when staining minichromosomemaintenance (MCM) proteins (Fig. 2d, e; anti-MCM5 staining is in H

MCM3AP Is Transcribed from a Promoter within an Intron of the Overlapping Gene for GANP

MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.

Chromosome Replication in Early Xenopus Embryos

Early embryos of X. laevis achieve exceptional rates of DNA replication and chromatin assembly. These processes have been studied by microinjecting DNA templates into eggs or by incubating DNA in egg extracts. The egg is able to regulate replication of injected DNA without requiring specialized DNA sequences. Assembly of DNA into the nucleosome subunits of chromatin involves interaction of DNA with complexes containing two classes of acidic protein, namely nucleoplasmin and N1/N2.