Table of Contents

Volume 1, Issue 5

  September/October 2010

  Pages 383 - 445

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EXTRA VIEWS

Open Access Article

Perspective on the metazoan nuclear pore complex

Tal Maimon and Ohad Medalia
Pages 383 - 386
http://dx.doi.org/10.4161/nucl.1.5.12332
Abstract | PDF

Fusing the inner and outer membranes of the nucleus, the nuclear pore complex (NPC) forms a selective portal which serves as the sole gateway of the nucleus. These aqueous translocation channels allow free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. Over the last several years major progress has been made in both structural determination of individual nucleopurins (Nups) and their complexes by X-ray crystallography and in structural analysis of the entire assembly by means of cryo-electron tomography. By combining cryo-electron tomography with advanced image processing techniques, the metazoan NPC structure from Xenopus oocytes was resolved to medium resolution, revealing novel details. Here, we discuss new features of the Xenopus NPC and consider future perspectives that will eventually allow resolution of the structure and function of NPCs with high accuracy.

Open Access Article

Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins

Roger Schneiter and Charles N. Cole
Pages 387 - 392
http://dx.doi.org/10.4161/nucl.1.5.12333
Abstract | PDF

The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

Open Access Article

GANP enhances the efficiency of mRNA nuclear export in mammalian cells

Vihandha O. Wickramasinghe, Murray Stewart and Ronald A. Laskey
Pages 393 - 396
http://dx.doi.org/10.4161/nucl.1.5.12351
Abstract | PDF

Nuclear export of mRNPs is mediated by transport factors such as NXF1 that bind mRNPs and mediate their translocation through the central channel of nuclear pores (NPC) using transient interactions with FG-nucleoporins. A number of nuclear factors enhance the efficiency of this process by concentrating mRNPs at the nuclear face of the pores. Although this enhancement has been explored mainly with the yeast TREX-2 complex, recent work has indicated that mammalian cells employ GANP (Germinal-centre Associated Nuclear Protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1-containing mRNPs to NPCs. GANP is constructed from several domains that show local homology to FGnucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. Whereas yeast TREX-2 is located primarily at nuclear pores, some GANP is located in the nuclear interior in addition to that found at the pores. GANP depletion inhibits bulk mRNA export, resulting in retention of mRNPs and NXF1 in punctate foci within the nucleoplasm, consistent with GANP’s being an integral component of the mammalian mRNA export machinery. Here, we discuss 2 the model for GANP function presented in our recent paper and its implications for the mechanism of mRNA export in mammalian cells.

Open Access Article

Multiple novel functions of Lamina associated polypeptide 2α in striated muscle

Ivana Gotic and Roland Foisner
Pages 397 - 401
http://dx.doi.org/10.4161/nucl.1.5.12394
Abstract | PDF

Lamina-associated polypeptide 2α (LAP2α) is a nucleoplasmic protein that interacts with A-type lamins and the retinoblastoma protein (pRb) and affects pRb-mediated cell cycle regulation and chromatin organization. Mutations in lamin A/C and LAP2α cause late onset striated muscle diseases, but the molecular mechanisms are poorly understood. We have recently reported on the striated muscle phenotype of LAP2α-deficient mice, revealing new unexpected roles of LAP2α. Loss of LAP2α in skeletal muscle caused an upregulated stem cell-type gene expression in muscle satellite cell progeny and their delayed myogenic differentiation in vitro. In vivo, the myofiber-associated muscle stem cell pool was increased. In addition, absence of LAP2α promoted muscle remodeling towards fast myofiber types in the soleus muscle of old animals. In cardiac tissue, deletion of LAP2α caused systolic dysfunction in young mice with an increased susceptibility for fibrosis in old animals. The functional impairment in the heart was accompanied by a deregulation of major cardiac transcription factors, GATA4 and MEF2c, and activation of compensatory pathways, including the downregulation of β-adrenergic receptor signaling. Here we discuss potential functions of LAP2α in striated muscle at molecular level and how loss of these functions may cause the diverse muscle phenotypes. We propose that LAP2α serves as a transcriptional co-regulator, which controls muscle specific gene expression during muscle regeneration, muscle remodeling and stress response.

Open Access Article

A role for DNA sequence in controlling the spatial organization of the genome

Sara Ahmed and Jason H. Brickner
Pages 402 - 406
http://dx.doi.org/10.4161/nucl.1.5.12637
Abstract | PDF

Recruitment of genes to the nuclear periphery upon transcriptional activation is a common phenomenon in Saccharomyces cerevisiae. We have recently identified DNA elements called gene recruitment sequences (GRSs) in the promoters of genes that are recruited to the nuclear periphery. These elements are necessary for peripheral targeting of genes. GRSs also function as DNA zip codes: they are sufficient to target an ectopic locus to the nuclear periphery. Targeting promotes full transcription and involves the interaction of promoters with the Nuclear Pore Complex (NPC). GRSs are widespread across the yeast genome, and are enriched in the promoters of genes induced by protein folding stress. Here, we place these observations in the context of the more global topic of genome organization and speculate about how the position of genes impacts their expression.

Open Access Article

LINCing lamin B2 to neuronal migration: growing evidence for cell-specific roles of B-types lamins

Catherine Coffinier, Loren G. Fong and Stephen G. Young
Pages 407 - 411
http://dx.doi.org/10.4161/nucl.1.5.12830
Abstract | PDF

Nuclear lamins are major components of the nuclear lamina, and play essential roles in supporting the nucleus and organizing nuclear structures. While a large number of clinically important mutations have been mapped to the LMNA gene in humans, very few mutations have been associated with the B-type lamins. We have shown that lamin B2-deficiency in mice results in severe brain abnormalities. While the early stages of forebrain development in lamin B2-deficient mice appear to be normal, cortical neurons fail to migrate and organize into proper layers within the cerebral cortex. The morphogenesis of the hippocampus and cerebellum is also severely impaired. These phenotypes are reminiscent of lissencephaly, a human brain developmental disorder characterized by an abnormal neuronal migration. Most mutations in lissencephaly patients affect cytoplasmic regulators of nuclear translocation, which is a crucial step in neuronal migration. The phenotypes of lamin B2-deficient mice suggest that lamin B2 may also play a key role in nuclear translocation. Potential mechanisms for lamin B2 involvement, which include mechanical and non-mechanical roles, and participation in LINC complexes in the nuclear envelope, are discussed along with evidence that lamins B1 and B2 play distinct, cell-specific functions.




RESEARCH PAPERS

Open Access Article

Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20

Marta Pabis, Noa Neufeld, Yaron Shav-Tal and Karla Neugebauer
Pages 412 - 421
http://dx.doi.org/10.4161/nucl.1.5.12839
Abstract | PDF

The nuclear cap-binding complex (CBC) is a heterodimer composed of CBP20 and CBP80 subunits and has roles in the biogenesis of messenger RNAs (mRNAs), small nuclear RNAs (snRNAs) and microRNAs. CBP20 is a phylogenetically conserved protein that interacts with the 7-methyl guanosine (m⁷G) cap added to the 5' end of all RNA polymerase II transcripts. CBP80 ensures high affinity binding of the cap by CBP20 and provides a platform for interactions with other factors. Here we characterize an alternative splice variant of CBP20, termed CBP20S. The CBP20S transcript has an in-frame deletion, leading to the translation of a protein lacking most of the RNA recognition motif (RRM). We show that CBP20S is conserved among mammalian species and is expressed in human cell lines and bone marrow cells. Unlike the full-length CBP20, CBP20S does not bind CBP80 or the m⁷G cap. Nevertheless, CBP20S does bind mRNA, is localized to an active transcription site, and redistributed to nucleolar caps upon transcription inhibition. Our results suggest that this novel form CBP20S plays roles in transcription and/or RNA processing independent of CBP80 or the cap.

Open Access Article

Transportin 3 and importin α are required for effective nuclear import of HIV-1 integrase in virus-infected cells

Aviad Levin, Zvi Hayouka, Assaf Friedler and Abraham Loyter
Pages 422 - 431
http://dx.doi.org/10.4161/nucl.1.5.12903
Abstract | PDF

Unlike other retroviruses, Human immunodeficiency virus type-1 (HIV-1) can infect terminally differentiated cells, due to the ability of its pre-integration complex (PIC) to translocate via the host nuclear pore complex (NPC). The PIC Nuclear import has been suggested to be mediated by the viral integrase protein (IN), via either the importin α or transportin 3 (TNPO3/transportin-SR2) pathways.

We show that in virus-infected cells, IN interacts with both importin α and TNPO3, simultaneously or separately, suggesting a multiple use of nuclear import pathways. Disruption of either the IN-importin α or IN-TNPO3 complexes in virus-infected cells by specific cell-permeable-peptides resulted in inhibition of IN and viral cDNA nuclear import. Here we show that peptides which disrupt either one of these complexes block virus infection, indicating involvement of both pathways in efficient viral replication. Formation of IN-importin α and IN-TNPO3 complexes has also been observed in IN-transfected cultured cells. Using specific peptides, we demonstrate that in transfected cells but not in virus infected cells the importin α pathway overrides that of TNPO3. The IN-importin α and IN-TNPO3 complexes were not observed in virus-infected Rev-expressing cells, indicating the Rev protein's ability to disrupt both complexes.

Our work suggests that IN nuclear import requires the involvement of both importin α and TNPO3. The ability to inhibit nuclear import of the IN-DNA complex and consequently, virus infection by peptides that interrupt IN's interaction with either importin α or TNPO3 indicates that for efficient infection, nuclear import of IN should be mediated by both nuclear-import receptors.

Open Access Article

Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome

Yuexia Wang, Cecilia Östlund and Howard Worman
Pages 432 - 439
http://dx.doi.org/10.4161/nucl.1.5.12972
Abstract | PDF

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.




COMMENTARIES

Open Access Article

Estrogen fueled, nuclear Kiss:  Did it move for you?

Andrew S. Belmont
Pages 440 - 443
http://dx.doi.org/10.4161/nucl.1.5.13051
Abstract | PDF

A paper appearing in late 2008 attracted considerable attention with its description of a dramatic  juxtaposition of two estrogen responsive genes on different chromosomes within 15-60 minutes of adding estradiol.  These results challenged a growing consensus of limited chromosome mobility within interphase nuclei, while raising questions of whether a hitherto unknown molecular mechanism might exist to move chromosomes long distances within the nucleus.  These results also raised the fascinating question of how two genes on widely separated chromosomes might find each other over such a short time span.  Now, a more recent paper reports no such long-range interaction or chromosome movements  in the same cell types under what appear to be well replicated conditions, forcing a reexamination of the prior results.

Open Access Article

Cytologically "compact" nuclear domains: Reconsidering the nucleolus

Thoru Pederson
Pages 444 - 445
http://dx.doi.org/10.4161/nucl.1.5.13056
Abstract

The nucleolus is among the most striking cell components when viewed by phase contrast microscopy and yet, previous studies have revealed it to possess a lower molecular density than anticipated.  A surprising new report demonstrates that the nucleolus accommodates the assembly of a virus, this amidst building ribosomes and performing other host cell functions.