Active nuclear import and cytoplasmic retention of activation-induced deaminase

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

Nuclear trafficking and functions of endocytic proteins implicated in oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin-mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.

Structural basis for the nuclear import of the human androgen receptor

Ligand-dependent nuclear import is crucial for the function of the androgen receptor (AR) in both health and disease. The unliganded AR is retained in the cytoplasm but, on binding 5alpha-dihydrotestosterone, it translocates into the nucleus and alters transcription of its target genes. Nuclear import of AR is mediated by the nuclear import factor importin-alpha, which functions as a receptor that recognises and binds to specific nuclear localisation signal (NLS) motifs on cargo proteins. We show here that the AR binds to importin-alpha directly, albeit more weakly than the NLS of SV40 or nucleoplasmin. We describe the 2.6-angstroms-resolution crystal structure of the importin-alpha-AR-NLS complex, and show that the AR binds to the major NLS-binding site on importin-alpha in a manner different from most other NLSs. Finally, we have shown that pathological mutations within the NLS of AR that are associated with prostate cancer and androgen-insensitivity syndrome reduce the binding affinity to importin-alpha and, subsequently, retard nuclear import; surprisingly, however, the transcriptional activity of these mutants varies widely. Thus, in addition to its function in the nuclear import of AR, the NLS in the hinge region of AR has a separate, quite distinct role on transactivation, which becomes apparent once nuclear import has been achieved.

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

Nuclear import of HIV-1 integrase is inhibited in vitro by styrylquinoline derivatives

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

The effects of osmotic stress on the structure and function of the cell nucleus.

Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis.

Acto-myosin contractility rotates the cell nucleus

The nucleus of the eukaryotic cell functions amidst active cytoskeletal �laments, but its response to the stresses carried by these �laments is largely unexplored. We report here the results of studies of the translational and rotational dynamics of the nuclei of single �broblast cells, with the e�ects of cell migration suppressed by plating onto �bronectin-coated micro-fabricated patterns. Patterns of the same area but di�erent shapes and/or aspect ratio were used to study the e�ect of cell geometry on the dynamics. On circles, squares and equilateral triangles, the nucleus undergoes persistent rotational motion, while on high-aspect-ratio rectangles of the same area it moves only back and forth. The circle and the triangle showed respectively the largest and the smallest angular speed. We show that our observations can be understood through a hydrodynamic approach in which the nucleus is treated as a highly viscous inclusion residing in a less viscous uid of orientable �laments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and persistence time of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic ow around the nucleus, with pro�le and magnitude consistent with the results of our theoretical approach. Coherent intracellular ows and consequent nuclear rotation thus appear to be a generic property that cells must balance by speci�c mechanisms in order to maintain nuclear homeostasis

Actomyosin contractility rotates the cell nucleus

The cell nucleus functions amidst active cytoskeletal filaments, but its response to their contractile stresses is largely unexplored. We study the dynamics of the nuclei of single fibroblasts, with cell migration suppressed by plating onto micro-fabricated patterns. We find the nucleus undergoes noisy but coherent rotational motion. We account for this observation through a hydrodynamic approach, treating the nucleus as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and coherence of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic flow around the nucleus, with profile and magnitude consistent with the results of our theoretical approach. Coherent intracellular flows and consequent nuclear rotation thus appear to be an intrinsic property of cells.

Molecular analysis of maltotriose active transport and fermentation by Saccharomyces cerevisiae reveals a determinant role for the AGT1 permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and AM inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high-and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K-m, 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

一篇探索真核细胞核起源的力作——读李靖炎先生的长篇近作“The primitive nucleus model and the origin of the cell nucleus”

《胞内共生与细胞研究》( 《Endocytobiosis and Cell Research》)是由国际胞内共生学会(International Society of Endocytobiology, ISE)主办的期刊, 它主要发表有关内共生物(endosy mbioses)和真核细胞的起源、发展、分化、进化和系统发育的研究论文. 在胞内共生和真核细胞的起源进化研究领域享有声誉. 去年该杂志发表了我国学者李靖炎先生的长篇论著“The primitive nucleus model and the origin of the cell nucleus(原始细胞核的模型与细胞核的起源)”(见1999, 13(1-3):1-86). 国外一家 SCI 收录的专业性刊物为中国学者发表一篇长达86页的 Review, 实不多见, 是我国学者在此领域的殊荣。