Changes In Subcellular Localization Research Articles

Importance of Dimer Formation of Myocardin Family Members in the Regulation of Their Nuclear Export

Myocardin (Mycd) family members function as a transcriptional cofactor for serum response factor (SRF). Dimer formation is necessary to exhibit their function, and the coiled-coil domain (CC) plays a critical role in their dimerization. We have recently revealed a detailed molecular mechanism for their Crm1 (exportin1)-mediated nuclear export. Here, we found other unique significances of the dimerization of Mycd family members. Introduction of mutations in the CC of myocardin-related transcription factor A (MRTF-A) and truncated Mycd resulted in significant decreases in their cytoplasmic localization and increases in their nuclear localization. In accordance with such subcellular localization changes, their binding to Crm1 were reduced. These results indicate that the dimerization of Mycd family members is necessary for their Crm1-mediated nuclear export. We have recently found that the N-terminal region of Mycd consisting of 128 amino acids (Mycd N128) self-associates to Mycd via the central basic domain (CB), resulting in masking the Crm1-binding site. Such self-association of MRTF-A would be unlikely. In this study, we also revealed that the dimerization of Mycd was also necessary for this self-association. Wild-type Mycd activated SRF-mediated transcription more potently than Mycd lacking the Mycd N128 (Mycd ΔN128) did. These results suggest two possible functions of the Mycd N128: 1) stabilization of Mycd dimer to enhance SRF-mediated transcription and 2) positive regulation of the transactivation ability of Mycd. These findings provide a new insight into the functional regulation of Mycd family members.

Changes in subcellular localization reveal interactions between human cytomegalovirus terminase subunits

BackgroundDuring herpesvirus replication, terminase packages viral DNA into capsids. The subunits of herpes simplex virus terminase, UL15, UL28, and UL33, assemble in the cytoplasm prior to nuclear import of the complex.MethodsTo detect similar interactions between human cytomegalovirus terminase subunits, the orthologous proteins UL89, UL56, and UL51 were expressed in HEK-293 T cells (via transfection) or insect cells (via baculovirus infection) and subcellular localizations were detected by cellular fractionation and confocal microscopy.ResultsIn both cell types, UL56 and UL89 expressed alone were exclusively cytoplasmic, whereas UL51 was ~50% nuclear. Both UL89 and UL56 became ~50% nuclear when expressed together, as did UL56 when expressed with UL51. Nuclear localization of each protein was greatest when all three proteins were co-expressed.ConclusionsThese results support inclusion of UL51 as an HCMV terminase subunit and suggest that nuclear import of human cytomegalovirus terminase may involve nuclear import signals that form cooperatively upon subunit associations.

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3. Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN-FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t-GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1-N-naphthylphthalamic acid (NPA) treatment. The tissue-specific expression patterns of OsPIN3t were also investigated using a β-glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild-type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.

Pur‐alpha regulates RhoA developmental expression and downstream signaling

Pur-alpha is an essential protein for postnatal brain development which localizes specifically to dendrites where it plays a role in the translation of neuronal RNA. Mice lacking Pur-alpha display decreased neuronogenesis and impaired neuronal differentiation. Here we examined two Rho GTPases, Rac1 and RhoA, which play opposing roles in neurite outgrowth and are critical for dendritic maturation during mouse brain development in the presence and absence of Pur-alpha. Pur-alpha is developmentally regulated in the mouse brain with expression beginning shortly after birth and rapidly increasing to peak during the third week of postnatal development. RhoA levels analyzed by Western blotting rapidly fluctuated in the wild-type mouse brain, however, in the absence of Pur-alpha, a decrease in RhoA levels shortly after birth and a delay in the cycling of RhoA regulation was observed leading to reduced basal levels of RhoA after day 10 postnatal. Immunohistochemistry of brain tissues displayed reduced RhoA levels in the cortex and cerebellum and loss of perinuclear cytoplasmic labeling of RhoA within the cortex in the knockout mouse brain. While Rac1 levels remained relatively stable at all time points during development and were similar in both wild-type and Pur-alpha knockout mice, changes in subcellular localization of Rac1 were seen in the absence of Pur-alpha. These findings suggest that Pur-alpha can regulate RhoA at multiple levels including basal protein levels, subcellular compartmentalization, as well as turnover of active RhoA in order to promote dendritic maturation.

185P - Activation of Akt by Hypoxia Predicts Poor Outcome in Malignant Pleural Mesothelioma (MPM)

ABSTRACT Introduction The Phosphatidylinositol-3 kinase (PI3K)/Akt (PKB) pathway is activated in a wide range of tumour types, and plays a central role in cell survival. Recent evidence indicates that hypoxia induces upregulation of Akt and activation of the Akt/PKB pathway. Once activated, Akt phosphorylates a variety of downstream substrates, including FOXO3a, which decreases transcription of the pro-apoptotic factor Bim, facilitating cell survival. We have shown that nuclear phospho-Akt (pAkt) expression is associated with more advanced disease or poor prognosis in Non-Small Cell Lung Cancer and MPM. Carbonic Anhydrase (CA)-IX, a tumour-specific member of the carbonic anhydrase family, is a surrogate marker of hypoxia overexpressed in solid tumours. This study examines the expression of CA-IX and phosphorylated Akt (pAkt) in tumour samples from patients with MPM, correlating expression with established prognostic factors. The role of pAkt in the survival of MPM cell lines exposed to both normoxia and hypoxia was also examined with both PI3K and PI3k-mTOR inhibitors. Methods Tumour sections were stained using pAkt and CA-IX specific antibodies. The effect of hypoxia on pAkt, Akt and Bim expression in 4 MPM cell lines in the presence or absence of LY294002 (phosphatidylinositol-3-kinase inhibitor) was examined by Western blot. The percentage of apoptotic cells was also quantified in these cells using FACs and High-Content Analysis (HCA). Changes in subcellular localisation of pAkt and FOXO3a were quantified using HCA. The cells were also transfected with siRNAs to PDK1or DNA-PKcs (two kinases known to catalyse the phosphorylation of Akt) and the effect on pAkt expression was determined by Western blot. Results A positive association between CA-IX and pAkt staining implies intra-tumoural hypoxia may stimulate Akt phosphorylation. Multivariate analysis showed increased expression of nuclear pAkt was associated with poor survival. Hypoxia induced the activation of Akt in MPM cell lines resulting in changes in the subcellular localisation of pAkt and FOXO3a. A greater increase in the level of apoptosis was seen in cells treated with PI3K inhibitors under hypoxia. Conclusion pAkt plays an anti-apoptotic role in MPM and represents a potential therapeutic target particularly in hypoxic tumours. Disclosure All authors have declared no conflicts of interest.

Increase in phosphorylation of PDK1 and cell survival after acute spinal cord injury

3-Phosphoinositide‐dependent protein kinase-1 (PDK1), which phosphorylates and activates a group of kinases, plays important roles in cellular metabolism, growth, proliferation and survival. However, the functions of PDK1 in central nervous system (CNS) injury remain an enigma. To elucidate the expressions and possible functions of PDK1 and its phosphorylation in CNS injury and repair, we performed an acute spinal cord injury (SCI) model in adult rats and detected the expression and localization of serine-241 phosphorylated PDK1 (p-PDK1s241). Western blot and immunohistochemistry showed that serine-241 phosphorylated PDK1 (p-PDK1s241) started increasing by 6h after damage and peaked at 12h, then declined to basal levels by 3days after injury. Immunohistochemical staining also revealed subcellular localization changes of p-PDK1s241 staining between nucleus and cytoplasm including neurons and glial cells after injury. Double immunofluorescence labeling suggested that p-PDK1s241 primarily localized in neurons and oligodendrocytes. It might also be expressed in other glial cells of spinal cord tissues within 2mm from the epicenter at 12h post-injury. Moreover, double staining indicated that p-PDK1s241 and active caspase-3 showed different cellular distributions after SCI. Together with previous reports, we hypothesize that phosphorylation of PDK1 may be associated with cell survival and suggest PDK1 as a novel target for neuroprotection and functional repair in SCI.

Increase in phosphorylation of PDK1 and cell survival after acute spinal cord injury

3-Phosphoinositidedependent protein kinase-1 (PDK1), which phosphorylates and activates a group of kinases, plays important roles in cellular metabolism, growth, proliferation and survival. However, the functions of PDK1 in central nervous system (CNS) injury remain an enigma. To elucidate the expressions and possible functions of PDK1 and its phosphorylation in CNS injury and repair, we performed an acute spinal cord injury (SCI) model in adult rats and detected the expression and localization of serine-241 phosphorylated PDK1 (p-PDK1s241). Western blot and immunohistochemistry showed that serine-241 phosphorylated PDK1 (p-PDK1s241) started increasing by 6h after damage and peaked at 12h, then declined to basal levels by 3days after injury. Immunohistochemical staining also revealed subcellular localization changes of p-PDK1s241 staining between nucleus and cytoplasm after injury including neurons and glial cells. Double immunofluorescence labeling suggested that p-PDK1s241 primarily localizes in neurons and oligodendrocytes. It might also be expressed in other glial cells of spinal cord tissues within 2mm from the epicenter at 12h post-injury. Moreover, double staining indicated that p-PDK1s241 and active caspase-3 showed different cellular distributions after SCI. Together with previous reports, we hypothesize that phosphorylation of PDK1 may be associated with cell survival and suggest PDK1 as a novel target for neuroprotection and functional repair in SCI.

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.

Identification of protein SUMOylation sites by mass spectrometry using combined microwave‐assisted aspartic acid cleavage and tryptic digestion

Small-ubiquitin-like modifier (SUMO) is a posttranslational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization and mitosis, among other cellular processes, are known to occur. However, although the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly "tag" that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32-amino-acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches, which serve to shorten the SUMO tag and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-time-of-flight (TOF) and LC/MS instrumentation, along with collision induced dissociation (CID) and electron transfer dissociation (ETD).

β‐2 Spectrin is involved in hepatocyte proliferation through the interaction of TGFβ/Smad and PI3K/AKT signalling

Transforming growth factor (TGF) β signalling pathway plays a crucial role in liver regeneration following partial hepatectomy in mice. Evidence demonstrated that β-2 Spectrin is involved in TGFβ/Smad signalling pathway as a Smad3/4 adaptor protein. The aim of this study was to explore the role of β-2 Spectrin in hepatocyte proliferation. β-2 Spectrin expression was evaluated in mice receiving partial hepatectomy. The effect of siRNA against β-2 Spectrin on hepatocyte proliferation was determined. The interaction between TGFβ/Smad and PI3K/Akt signalling was investigated. Hepatic β-2 Spectrin decreased dramatically 2 days after 70% hepatectomy in mice. In AML-12 cells, hepatocyte proliferation was inhibited after the stimulation of TGF β1 and a reduction in β-2 Spectrin mediated by siRNA resulted in increase in proliferative response. Confocal results revealed that β-2 Spectrin represented a key regulator in TGFβ/Smad signalling through controlling Smad3/4 subcellular localization. Moreover, Alternation of Akt phosphorylation led to the change in subcellular localization of Smad2, 3, 4 and β-2 Spectrin, A reduction in Smad2, 3 and 4 mediated by siRNA resulted in the induction of pAkt expression. These findings reveal that β-2 Spectrin plays a crucial role in hepatocyte proliferation, which contributes to liver regeneration following hepatectomy in mice. In addition, PI3K/Akt is involved in TGFβ/Smad signalling pathway through the interaction with Smad proteins and β-2 Spectrin.

DNA Damage Response: Multilevel Proteomics Gains Momentum

In this issue of Molecular Cell, Beli et al. (2012) introduce a multilevel proteomics approach for parallel quantification of protein phosphorylation, acetylation, and abundance and apply this to the complex signaling network of the DNA damage response.

Physicians and Advanced Practice Registered Nurses: Working Together As a Team

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth and development. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the *Arabidopsis thaliana* root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK1α1 catalytic subunit is enriched in the nuclei of root meristematic cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a *snrk2.2 snrk2.3* double mutant causes constitutive cytoplasmic localization of SnRK1α1 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK1α1 in the nucleus. In response to elevated ABA levels, SnRK1α1 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1α1 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK1α1 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK1α1 nuclear exit is a premise for this repression. Finally, the SnRK2-dependent changes in SnRK1α1 subcellular localization are specific to the proliferation zone of the meristem, underscoring the relevance of this mechanism for growth regulation.

Spatiotemporal Expression of Testicular Protein Kinase 1 After Rat Sciatic Nerve Injury

Testicular protein kinase 1 (TESK1), a serine/threonine kinase, has been found expressing in various tissues and cell lines. Previous reports have shown that TESK1 plays an important role in regulating actin reorganization of spreading cell on fibronectin via phosphorylating cofilin. Because of the importance of actin reorganization in radial sorting and remyelination of peripheral nerve regeneration, we investigated the spatiotemporal expression of TESK1 in a rat sciatic nerve crush model. We observed that sciatic nerve crush resulted in a significant upregulation of TESK1 from 5 days to 2 weeks and subsequent return to the control level at 4 weeks. At its peak expression, TESK1 expressed mainly in both Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, upregulation of TESK1 was approximately in parallel with Oct-6, and numerous SCs expressing TESK1 were Oct-6 positive. Experiments with Schwann cell primary cultures revealed that TESK1 accumulated at F-actin-rich lamellipodia of the cell periphery when SCs were plated on fibronectin, whereas it was distributed in the cytoplasm in the case of non-stimulated cells. Thus, these findings suggest that TESK1 plays important roles in promyelinating SCs, potentially through subcellular localization change and participation in integrin-mediated actin reorganization.

Hsp90 and its co-chaperone, Sgt1, as autoantigens in dilated cardiomyopathy

Recently, it has been suggested that some heat shock proteins such as Hsp70 and Hsp60 are involved in autoimmune diseases including cardiospecific ones. In this work we focused on the involvement of another wellknown heat shock protein, Hsp90, and its novel co-chaperone,Sgt1, in dilated cardiomyopathy (DCM). We found that the level of autoantibodies against these two proteins was significantly higher in patients with DCM and ischemic heart disease than in sera of healthy donors. We have also analyzed the expression level and subcellular localization of Hsp90 and Sgt1 in diseased myocardia. Using Western blot we found changes in subcellular localization of Hsp90 in the left ventricle of DCM hearts while the total level of this protein remained unchanged. Regarding the Sgt1 protein, we found an increased level in DCM and no changes in subcellular localization. Taken together, our data suggest that Hsp90 and Sgt1 might be involved in the progression of heart failure and might serve as markers for cardiomyopathies of different origin.

Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

Myotubular myopathy and the neuromuscular junction: a novel therapeutic approach from mouse models

SUMMARYMyotubular myopathy (MTM) is a severe congenital muscle disease characterized by profound weakness, early respiratory failure and premature lethality. MTM is defined by muscle biopsy findings that include centralized nuclei and disorganization of perinuclear organelles. No treatments currently exist for MTM. We hypothesized that aberrant neuromuscular junction (NMJ) transmission is an important and potentially treatable aspect of the disease pathogenesis. We tested this hypothesis in two murine models of MTM. In both models we uncovered evidence of a disorder of NMJ transmission: fatigable weakness, improved strength with neostigmine, and electrodecrement with repetitive nerve stimulation. Histopathological analysis revealed abnormalities in the organization, appearance and size of individual NMJs, abnormalities that correlated with changes in acetylcholine receptor gene expression and subcellular localization. We additionally determined the ability of pyridostigmine, an acetylcholinesterase inhibitor, to ameliorate aspects of the behavioral phenotype related to NMJ dysfunction. Pyridostigmine treatment resulted in significant improvement in fatigable weakness and treadmill endurance. In all, these results describe a newly identified pathological abnormality in MTM, and uncover a potential disease-modifying therapy for this devastating disorder.

A moderate decline in U937 cell GSH levels triggers PI3 kinase/Akt-dependent Bad phosphorylation, thereby preventing an otherwise prompt apoptotic response

We report that a moderate decline in GSH levels causes remarkable changes in Bad sub-cellular localization. An about 30% reduction of the GSH pool, regardless of whether mediated by diamide or dl-buthionine-[S,R]-sulfoximine, indeed promoted loss of the fraction of Bad normally associated with the mitochondria of untreated U937 cells via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Interestingly, inhibition of this pathway was associated with an unexpected delayed lethal response, preceded by the translocation and enforced accumulation of Bad and Bax in the mitochondrial compartment, prevented by inhibitors of mitochondrial permeability transition and characterized by morphological and biochemical features of apoptosis. Collectively, the results herein presented demonstrate that mild redox imbalance associated with a slight reduction of the GSH pool commits U937 cells to apoptosis, however prevented by events leading to PI3K/Akt-dependent mitochondrial loss of Bad.

Concentration‐dependent transitions govern the subcellular localization of islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic β-cells. In type II diabetes, IAPP aggregates in a process that is associated with β-cell dysfunction and loss of β-cell mass. The relationship between IAPP's conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α-helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPP's capacity to adopt aggregated membrane-bound α-helical, and not β-sheet, states. Our findings suggest that upon α-helical mediated oligomerization, IAPP acquires cell-penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.