Key Modulators of the Stress Granule Response TIA1, TDP-43, and G3BP1 Are Altered by Polyglutamine-Expanded ATXN7.

Spinocerebellar ataxia type 7 (SCA7) and other polyglutamine (polyQ) diseases are caused by expansions of polyQ repeats in disease-specific proteins. Aggregation of the polyQ proteins resulting in various forms of cellular stress, that could induce the stress granule (SG) response, is believed to be a common pathological mechanism in these disorders. SGs can contribute to cell survival but have also been suggested to exacerbate disease pathology by seeding protein aggregation. In this study, we show that two SG-related proteins, TDP-43 and TIA1, are sequestered into the aggregates formed by polyQ-expanded ATXN7 in SCA7 cells. Interestingly, mutant ATXN7 also localises to induced SGs, and this association altered the shape of the SGs. In spite of this, neither the ability to induce nor to disassemble SGs, in response to arsenite stress induction or relief, was affected in SCA7 cells. Moreover, we could not observe any change in the number of ATXN7 aggregates per cell following SG induction, although a small, non-significant, increase in total aggregated ATXN7 material could be detected using filter trap. However, mutant ATXN7 expression in itself increased the speckling of the SG-nucleating protein G3BP1 and the SG response. Taken together, our results indicate that the SG response is induced, and although some key modulators of SGs show altered behaviour, the dynamics of SGs appear normal in the presence of mutant ATXN7.

Polyglutamine expanded Ataxin-7 induces DNA damage and alters FUS localization and function.

Polyglutamine (polyQ) diseases, such as Spinocerebellar ataxia type 7 (SCA7), are caused by expansions of polyQ repeats in disease specific proteins. The sequestration of vital proteins into aggregates formed by polyQ proteins is believed to be a common pathological mechanism in these disorders. The RNA-binding protein FUS has been observed in polyQ aggregates, though if disruption of this protein plays a role in the neuronal dysfunction in SCA7 or other polyQ diseases remains unclear. We therefore analysed FUS localisation and function in a stable inducible PC12 cell model expressing the SCA7 polyQ protein ATXN7. We found that there was a high degree of FUS sequestration, which was associated with a more cytoplasmic FUS localisation, as well as a decreased expression of FUS regulated mRNAs. In contrast, the role of FUS in the formation of γH2AX positive DNA damage foci was unaffected. In fact, a statistical increase in the number of γH2AX foci, as well as an increased trend of single and double strand DNA breaks, detected by comet assay, could be observed in mutant ATXN7 cells. These results were further corroborated by a clear trend towards increased DNA damage in SCA7 patient fibroblasts. Our findings suggest that both alterations in the RNA regulatory functions of FUS, and increased DNA damage, may contribute to the pathology of SCA7.

Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein Samp1.

We have investigated a possible role for the inner nuclear membrane protein Samp1 (also known as TMEM201) in the mitotic machinery. Live-cell imaging showed that Samp1a-YFP (Samp1a is the short isoform of Samp1) distributed as filamentous structures in the mitotic spindle, partially colocalising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistent with this, mitotic spindles in Samp1-depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes that were rescued by overexpression of Samp1a-YFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and the HAUS6 subunit of the Augmin complex in live cells. The levels of HAUS6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of HAUS6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.

MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells.

Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.

Samp1 is functionally associated with the LINC complex and A-type lamina networks.

The transmembrane inner nuclear membrane (INM) protein Samp1 is required for anchoring centrosomes near the nuclei. Using high-resolution fluorescence microscopy we show that Samp1 is distributed in a distinct and characteristic pattern in the nuclear envelope (NE), where it partially colocalizes with the LINC complex protein Sun1. By studying the localization of Samp1 deletion mutants and fusion proteins, we conclude that the cysteine-rich N-terminal half of Samp1 is nucleoplasmically exposed and is responsible for targeting to the INM. It contains four conserved CxxC motifs with the potential to form zinc fingers. The distribution of cysteine-to-alanine substitution mutants, designed to prevent zinc finger formation, showed that NE localization of Samp1 depends on intact CxxC motifs. Overexpression of Samp1 zinc finger mutants produced an abnormal dominant phenotype characterized by disrupted organization of a selective subset NE proteins, including emerin, Sun1, endogenous Samp1 and, in some cases, lamin A/C, but not lamin B, Sun2 or nucleoporins. Silencing of Samp1 expression showed that emerin depends on Samp1 for its correct localization in the NE. Our results demonstrate that Samp1 is functionally associated with the LINC complex protein Sun1 and proteins of the A-type lamina network.

Anchored FRET sensors detect local caspase activation prior to neuronal degeneration.

BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-ß peptide as a causative agent in AD. RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-ß peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed. CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-ß peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

A transmembrane inner nuclear membrane protein in the mitotic spindle.

We have recently characterized a novel transmembrane protein of the inner nuclear membrane of mammalian cells. The protein has two very interesting features. First, despite being an integral membrane protein it is able to concentrate in the membranes colocalizing with the mitotic spindle in metaphase and anaphase. Hence, the protein was named Samp1, Spindle associated membrane protein 1. Secondly, it displays a functional connection to centrosomes. This article discusses various aspects of Samp1 in relation to possible cellular function(s).

An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells.

Here, we characterize a transmembrane protein of the nuclear envelope that we name spindle-associated membrane protein 1 (Samp1). The protein is conserved in metazoa and fission yeast and is homologous to Net5 in rat and Ima1 in Schizosaccharomyces pombe. We show that, in human cells, the protein is a membrane-spanning polypeptide with an apparent molecular mass of 43 kDa. This is consistent with a predicted polypeptide of 392 amino acids that has five transmembrane segments and its C-terminus exposed to the nucleoplasm. During interphase, Samp1 was specifically distributed in the inner nuclear membrane. Post-transcriptional silencing of Samp1 expression resulted in separation of centrosomes from the nuclear envelope, indicating that it is functionally connected to the cytoskeleton. At the onset of mitosis, most of the protein dispersed out into the ER, as expected. However, during mitosis, a significant fraction of the protein specifically localized to the polar regions of the mitotic spindle. We demonstrate for the first time, in human cells, the existence of a membranous structure overlapping with the mitotic spindle. Interestingly, another integral inner nuclear membrane protein, emerin, was absent from the spindle-associated membranes. Thus, Samp1 defines a specific membrane domain associated with the mitotic spindle.

Targeting cytokine expression in glial cells by cellular delivery of an NF-kappaB decoy.

Inhibition of nuclear factor (NF)-kappaB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer's disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-kappaB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer beta-amyloid peptide in the presence of the inflammatory cytokine interleukin (IL)-1beta. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-kappaB binding activity and IL-6 mRNA expression, respectively.

Dynamic properties of nuclear pore complex proteins in gp210 deficient cells.

Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.

Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels.

c-Myc is a predominantly nuclear transcription factor that is a substrate for rapid turnover by the proteasome system. Cancer-related mutations in c-Myc lead to defects in its degradation and thereby contribute to the increase in its cellular level that is associated with the disease. Little is known about the mechanisms that target c-Myc to the proteasomes. By using a GFP fusion protein and live analysis we show that c-Myc shuttles between the nucleus and cytoplasm and thus it could be degraded in either compartment. Strikingly, at elevated levels of expression c-Myc accumulates at nucleoli in some cells, consistent with saturation of a nucleolus-associated degradation system in these cells. This idea is further supported by the observation that proteasome inhibitor treatment causes accumulation of c-Myc at the nucleoli of essentially all cells. Under these conditions c-Myc is relatively stably associated with the nucleolus, as would be expected if the nucleolus functions as a sequestration/degradation site for excess c-Myc. Furthermore, during elevated c-Myc expression or proteasome inhibition, nucleoli that are associated with c-Myc also accumulate proteasomes. c-Myc and proteasomes co-localise in intranucleolar regions distinct from the dense fibrillar component of the nucleolus. Based on these results we propose a model for c-Myc downregulation where c-Myc is sequestered at the nucleoli. Sequestration of c-Myc is accompanied by recruitment of proteasomes and may lead to subsequent degradation.

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.