Specific Cellular Compartments Research Articles

Protein kinase C-α and the regulation of diverse cell responses.

Protein kinase C (PKC) comprises a family of lipid-sensitive enzymes that have been involved in a broad range of cellular functions. PKC-α is a member of classical PKC with ubiquitous expression and different cellular localization. This unique PKC isoform is activated by various signals which evoke lipid hydrolysis, after activation it interacts with various adapter proteins and is localized to specific cellular compartments where it is devised to work. The universal expression and activation by various stimuli make it a perfect player in uncountable cellular functions including differentiation, proliferation, apoptosis, cellular transformation, motility, adhesion and so on. However, these functions are not intrinsic properties of PKC-α, but depend on cell types and conditions. The activities of PKC-α are managed by the various pharmacological activators/inhibitors and antisense oligonucleotides. The aim of this review is to elaborate the structural feature, and provide an insight into the mechanism of PKC-α activation and regulation of its key biological functions in different cellular compartments to develop an effective pharmacological approach to regulate the PKC-α signal array.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells.

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

Plant storage proteins - the main nourisching products - from biosynthesis to cellular storage depots.

Storage proteins of legumes are one of the main components of the human and animal diet. The substances collected in their seeds have the pro-health values, supporting the prevention of many civilization diseases. However, there are still many uncertainties about the mechanisms leading to the production of nutritious seeds. It is also difficult to identify which of their constituents and in what final form are responsible for the observed protective effects in vivo. In this work, on the background of different types of storage proteins, these deposited mainly in legumes were in the focus of interest. They were characterized on the example of pea (Pisum sativum) proteins. Mechanisms associated with their biosynthesis and transport to specific cellular compartments was presented. Ways of their post-translational processing, segregation and storage in the specific vacuoles were also discussed. Therefore, the paper presents the state-of-the-art knowledge concerning the processes making the accumulated protein deposits ready to use by plants, animals and humans.

Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.

LRRK2: from kinase to GTPase to microtubules and back.

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are intimately linked to both familial and sporadic Parkinson's disease. LRRK2 is a large protein kinase able to bind and hydrolyse GTP. A wealth of in vitro studies have established that the distinct pathogenic LRRK2 mutants differentially affect those enzymatic activities, either causing an increase in kinase activity without altering GTP binding/GTP hydrolysis, or displaying no change in kinase activity but increased GTP binding/decreased GTP hydrolysis. Importantly, recent studies have shown that all pathogenic LRRK2 mutants display increased kinase activity towards select kinase substrates when analysed in intact cells. To understand those apparently discrepant results, better insight into the cellular role(s) of normal and pathogenic LRRK2 is crucial. Various studies indicate that LRRK2 regulates numerous intracellular vesicular trafficking pathways, but the mechanism(s) by which the distinct pathogenic mutants may equally interfere with such pathways has largely remained elusive. Here, we summarize the known alterations in the catalytic activities of the distinct pathogenic LRRK2 mutants and propose a testable working hypothesis by which the various mutants may affect membrane trafficking events in identical ways by culminating in increased phosphorylation of select substrate proteins known to be crucial for membrane trafficking between specific cellular compartments.

Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication

BackgroundThe essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication.MethodsInfluenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference.ResultsExogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell.ConclusionsThese results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.

The PDE4 cAMP-Specific Phosphodiesterases: Targets for Drugs with Antidepressant and Memory-Enhancing Action.

The PDE4 cyclic nucleotide phosphodiesterases are essential regulators of cAMP abundance in the CNS through their ability to regulate PKA activity, the phosphorylation of CREB, and other important elements of signal transduction. In pre-clinical models and in early-stage clinical trials, PDE4 inhibitors have been shown to have antidepressant and memory-enhancing activity. However, the development of clinically-useful PDE4 inhibitors for CNS disorders has been limited by variable efficacy and significant side effects. Recent structural studies have greatly enhanced our understanding of the molecular configuration of PDE4 enzymes, especially the "long" PDE4 isoforms that are abundant in the CNS. The new structural data provide a rationale for the development of a new generation of PDE4 inhibitors that specifically act on long PDE4 isoforms. These next generation PDE4 inhibitors may also be capable of targeting the interactions of select long forms with their "partner" proteins, such as RACK1, β-arrestin, and DISC1. They would therefore have the ability to affect cAMP levels in specific cellular compartments and target localized cellular functions, such as synaptic plasticity. These new agents might also be able to target PDE4 populations in select regions of the CNS that are implicated in learning and memory, affect, and cognition. Potential therapeutic uses of these agents could include affective disorders, memory enhancement, and neurogenesis.

Cellular functions of LRRK2 implicate vesicular trafficking pathways in Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, associated with Parkinson's disease, have been shown to affect intracellular trafficking pathways in a variety of cells and organisms. An emerging theme is that LRRK2 can bind to multiple membranous structures in cells, and several recent studies have suggested that the Rab family of small GTPases might be important in controlling the recruitment of LRRK2 to specific cellular compartments. Once localized to membranes, LRRK2 then influences downstream events, evidenced by changes in the autophagy-lysosome pathway. Here, I will discuss available evidence that supports or challenges this outline, with a specific emphasis on those aspects of LRRK2 function that have been controversial or remain to be fully clarified.

Role of post-transcriptional regulation of mRNA stability in renal pathophysiology: focus on chronic kidney disease.

Chronic kidney disease (CKD) represents an important public health problem. Its progression to end-stage renal disease is associated with increased morbidity and mortality. The determinants of renal function decline are not fully understood. Recent progress in the understanding of post-transcriptional regulation of mRNA stability has helped the identification of both the trans- and cis-acting elements of mRNA as potential markers and therapeutic targets for difficult-to-diagnose and -treat diseases, including CKDs such as diabetic nephropathy. Human antigen R (HuR), a trans-acting element of mRNA, is an RNA binding factor (RBF) best known for its ability to stabilize AU-rich-element-containing mRNAs. Deregulated HuR subcellular localization or expression occurs in a wide range of renal diseases, such as metabolic acidosis, ischemia, and fibrosis. Besides RBFs, recent evidence revealed that noncoding RNA, such as microRNA and long noncoding RNA, participates in regulating mRNA stability and that aberrant noncoding RNA expression accounts for many pathologic renal conditions. The goal of this review is to provide an overview of our current understanding of the post-transcriptional regulation of mRNA stability in renal pathophysiology and to offer perspectives for this class of diseases. We use examples of diverse renal diseases to illustrate different mRNA stability pathways in specific cellular compartments and discuss the roles and impacts of both the cis- and trans-activating factors on the regulation of mRNA stability in these diseases.-Feigerlová, E., Battaglia-Hsu, S.-F. Role of post-transcriptional regulation of mRNA stability in renal pathophysiology: focus on chronic kidney disease.

The Long Non-Coding RNAs in Neurodegenerative Diseases: Novel Mechanisms of Pathogenesis.

Long-non-coding RNAs (lncRNAs), RNA molecules longer than 200 nucleotides, have been involved in several biological processes and in a growing number of diseases, controlling gene transcription, pre-mRNA processing, the transport of mature mRNAs to specific cellular compartments, the regulation of mRNA stability, protein translation and turnover. The fundamental role of lncRNAs in central nervous system (CNS) is becoming increasingly evident. LncRNAs are abundantly expressed in mammalian CNS in a specific spatio-temporal manner allowing a quick response to environmental/molecular changes. This article reviews the biology and mechanisms of action of lncRNAs underlying their potential role in CNS and in some neurodegenerative diseases. an increasing number of studies report on lncRNAs involvement in different molecular mechanisms of gene expression modulation in CNS, from neural stem cell differentiation mainly by chromatin remodeling, to control of neuronal activities. More recently, lncRNAs have been implicated in neurodegenerative diseases, including Alzheimer's Disease, where the role of BACE1-AS lncRNA has been widely defined. BACE1-AS levels are up-regulated in AD brains where BACE1-AS acts by stabilizing BACE1 mRNA thereby increasing BACE1 protein content and Aβ42 formation. In Frontotemporal dementia and Amyotrophic lateral sclerosis the lncRNAs NEAT1_2 and MALAT1 co-localize at nuclear paraspeckles with TDP-43 and FUS proteins and their binding to TDP-43 is markedly increased in affected brains. In Parkinson's Disease the lncRNA UCHL1-AS1 acts by directly promoting translation of UCHL1 protein leading to perturbation of the ubiquitin-proteasome system. Different lncRNAs, such as HTT-AS, BDNF-AS and HAR1, were found to be dysregulated in their expression also in Huntington's Disease. In Fragile X syndrome (FXS) and Fragile X tremor/ataxia syndrome (FXTAS) patients, the presence of CGG repeats expansion alters the expression of the lncRNAs FMR1-AS1 and FMR6. Interestingly, they are expressed in peripheral blood leukocytes, suggesting these lncRNAs may represent biomarkers for FXS/FXTAS early detection and therapy. Finally, the identification of the antisense RNAs SCAANT1-AS and ATXN8OS in spinocerebellar ataxia 7 and 8, respectively, suggests that very different mechanisms of action driven by lncRNAs may trigger neurodegeneration in these disorders. The emerging role of lncRNAs in neurodegenerative diseases suggests that their dysregulation could trigger neuronal death via still unexplored RNA-based regulatory mechanisms which deserve further investigation. The evaluation of their diagnostic significance and therapeutic potential could also address the setting up of novel treatments in diseases where no cure is available to date.

Making sense of NAD+ subcellular localization

A fluorescent NAD+ biosensor targeted to specific cellular compartments detects local fluctuation in NAD+ concentration.

DIC image reconstruction using an energy minimization framework to visualize optical path length distribution.

Label-free microscopy techniques have numerous advantages such as low phototoxicity, simple setup and no need for fluorophores or other contrast materials. Despite their advantages, most label-free techniques cannot visualize specific cellular compartments or the location of proteins and the image formation limits quantitative evaluation. Differential interference contrast (DIC) is a qualitative microscopy technique that shows the optical path length differences within a specimen. We propose a variational framework for DIC image reconstruction. The proposed method largely outperforms state-of-the-art methods on synthetic, artificial and real tests and turns DIC microscopy into an automated high-content imaging tool. Image sets and the source code of the examined algorithms are made publicly available.

Abstract 948: Microenvironmental origin of circulating miRNAs in lung cancer

Abstract INTRODUCTION We reported the identification of diagnostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Circulating miRNAs are released into the bloodstream by different mechanisms such as passive leakage from broken cells or active secretion through microvesicles or protein complexes. MATERIAL and METHODS To evaluate the origin and the release of the 24 miRNAs we analyzed their expression by real-time PCR in different lung cell types including fibroblasts, hematopoietic, endothelial, bronchial epithelial (HBECs) and tumor cells and in the respective culture medium (CM) as well as in plasma samples from heavy smokers and patients. RESULTS and DISCUSSION The analysis of lung cancer plasma samples in patients showed that miR-106a, miR-140, miR-142-3p, miR-17, miR-660, miR-320, miR-197 and mir-28-3p were those significantly returning to a physiological level after surgery (p<0.05). On the other hand, mir-126 and mir-451 remained deregulated after tumor resection suggesting an “host-related” origin and likely indicating the persistence of a risk profile. To evaluate which lung cell type express these miRNAs we performed in situ hybridization and observed that mir-451 is specifically expressed in lung interstitial alveolar walls by cells of the fibro-monocytic infiltrate whereas mir-126 seems to be expressed by endothelial cells. The analysis of the 24-miRNAs composing the signature in the various lung cell types showed an increased expression in specific cellular components such as that of mir-16-17-19b-106-145 in fibroblasts, mir-126 in endothelial cells, mir-451 and mir-142-3p in hematopoietic cells. The analysis of miRNAs released in CM showed that miRNAs were secreted differently and independently from their cellular levels and that tumor cells were the less involved in this release. Interestingly, we identified miRNAs that were expressed and released from blood cells only such as mir-451, mir-133 and mir-142-3p. To better characterize the origin of plasma miRNAs, we isolated exosomes from CM of the different cell types. We detected the presence of the 24 miRNAs in exosomes and identified exosomal miRNAs that were secreted by specific cellular compartments (mir-101 from blood cells, mir-145 from fibroblasts and mir-126 from endothelial cells). Furthermore, by analyzing the exosome fraction of 29 plasma samples of 21 smokers and 8 patients we confirmed the presence of all the 24 miRNAs inside exosomes and comparing miRNAs content in plasma and in their respective exosome fraction we observed a good degree of correlation (Pearson's R = 0.89, p for Pearson's R <0.001) indicating that miRNAs expression in exosomes may reflect miRNA levels in total plasma. CONCLUSION Our findings on the origin of circulating miRNAs support the conclusion that plasma miRNAs are heterogeneous and are actively secreted inside exosomes by different cellular components and not specifically by tumor cells. Citation Format: Orazio Fortunato, Cristina Borzi, Davide Conte, Giovanni Centonze, Mattia Boeri, Carla Verri, Linda Calzolari, Ugo Pastorino, Gabriella Sozzi. Microenvironmental origin of circulating miRNAs in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 948.

Abstract 3913: Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells

Abstract Introduction: Delivery of a nanoparticle payload to a specific cellular compartment is a prized goal of cancer nanomedicine. The well-known SELEX (systematic evolution of ligands by exponential enrichment) process is often used to identify aptamers that localize to target cellular compartments, but such aptamers cannot be guaranteed to traffic an attached payload nanoparticle to the target as well. We hypothesized that to find an aptamer capable of trafficking a nanoparticle to a specific cellular compartment, the screened library must consist of aptamer-nanoparticle conjugates. We therefore designed a screening procedure we call “Conjugate-SELEX”. In this process, the aptamer library is conjugated to liposomal nanoparticles, creating a nanoparticle-aptamer conjugate library for screening. At each round of Conjugate-SELEX, aptamers recovered from the desired cellular compartment are amplified, and used to create the conjugate library for the next round of Conjugate-SELEX. Negative screens against off-target cells (e.g. hepatocytes, unwanted uptake in which, causes many of the toxicities associated with nanoparticle therapeutics) facilitate the identification of an aptamer that efficiently transports payload into target cells while minimizing the uptake in off-target cells. Using fluorescently tagged particles, imaging the cells at each stage verifies the successful transport of the payload particles into the cytosol. Methods: We used conjugate-SELEX to screen for aptamers that carried an attached nanoparticle payload to the cytosol of UM-SCC-22A oral cancer cell line, while not being taken up by THLE-3 hepatocytes. IonTorrent sequencing of the remaining aptamers after each round demonstrated convergence to a small family of sequences with only one base difference between members. Mass spectrometric proteomics identified the surface receptor bound by these sequences as the neuroblast differentiation-associated protein AHNAK (desmoyokin) a ubiquitous intracellular protein with surface expression exclusively in certain epithelial cell types. Uptake studies with concertedly synthesized hit aptamer sequences showed enhanced uptake of targeted nanoparticles over controls, and increased cytotoxicity of doxorubicin in the nanoparticles over controls. Conclusions: Conjugate-SELEX is an excellent means of identifying aptamers with the ability to transport attached nanoparticle payloads to targeted cellular compartments, while minimizing off target effects. The technique also lends itself to the identification of surface receptors mediating the trafficking.[D.G., A.A., and N.V. contributed equally to this work.] Citation Format: Qingshan Mu, Akshaya Annapragada, Mayank Srivastava, Varatharasa Thiviyanathan, Xin Li, David Gorenstein, Annanth Annapragada, Nadarajah Vigneswaran. Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3913.

Extent of pre-translational regulation for the control of nucleocytoplasmic protein localization.

BackgroundAppropriate protein subcellular localization is essential for proper cellular function. Central to the regulation of protein localization are protein targeting motifs, stretches of amino acids serving as guides for protein entry in a specific cellular compartment. While the use of protein targeting motifs is modulated in a post-translational manner, mainly by protein conformational changes and post-translational modifications, the presence of these motifs in proteins can also be regulated in a pre-translational manner. Here, we investigate the extent of pre-translational regulation of the main signals controlling nucleo-cytoplasmic traffic: the nuclear localization signal (NLS) and the nuclear export signal (NES).ResultsMotif databases and manual curation of the literature allowed the identification of 175 experimentally validated NLSs and 120 experimentally validated NESs in human. Following mapping onto annotated transcripts, these motifs were found to be modular, most (73 % for NLS and 88 % for NES) being encoded entirely in only one exon. The presence of a majority of these motifs is regulated in an alternative manner at the transcript level (61 % for NLS and 72 % for NES) while the remaining motifs are present in all coding isoforms of their encoding gene. NLSs and NESs are pre-translationally regulated using four main mechanisms: alternative transcription/translation initiation, alternative translation termination, alternative splicing of the exon encoding the motif and frameshift, the first two being by far the most prevalent mechanisms. Quantitative analysis of the presence of these motifs using RNA-seq data indicates that inclusion of these motifs can be regulated in a tissue-specific and a combinatorial manner, can be altered in disease states in a directed way and that alternative inclusion of these motifs is often used by proteins with diverse interactors and roles in diverse pathways, such as kinases.ConclusionsThe pre-translational regulation of the inclusion of protein targeting motifs is a prominent and tightly-regulated mechanism that adds another layer in the control of protein subcellular localization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2854-4) contains supplementary material, which is available to authorized users.

A bacterial phytochrome-based optogenetic system controllable with near-infrared light.

Light-mediated control of protein-protein interactions to regulate metabolic pathways is an important approach of optogenetics. Here, we report the first optogenetic system based on a reversible light-induced binding between a bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. We extensively characterized the BphP1–PpsR2 interaction both in vitro and in mammalian cells, and then used it to translocate target proteins to specific cellular compartments, such as plasma membrane and nucleus. Applying this approach we achieved a light-control of cell morphology resulting in the substantial increase of cell area. We next demonstrated the light-induced gene expression with the 40-fold contrast in cultured cells, 32-fold subcutaneously and 5.7-fold in deep tissues in mice. The unique characteristics of the BphP1–PpsR2 optogenetic system are its sensitivity to 740–780 nm near-infrared light, ability to utilize an endogenous biliverdin chromophore in eukaryotes including mammals, and spectral compatibility with blue-light optogenetic systems.

Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins.

Organisms from all kingdoms of life use iron-sulfur proteins (FeS-Ps) in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of FeS-Ps. Sixty-one percent of human FeS-Ps bind Fe4S4 clusters, whereas 39% bind Fe2S2 clusters. However, this relative ratio varies significantly depending on the specific cellular compartment. We compared the portfolio of human FeS-Ps to 12 other eukaryotes and to about 700 prokaryotes. The comparative analysis of the organization of the prokaryotic homologues of human FeS-Ps within operons allowed us to reconstruct the human functional networks involving the conserved FeS-Ps common to prokaryotes and eukaryotes. These functional networks have been maintained during evolution and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps. Purine metabolism is another process including several FeS-Ps, in which BOLA proteins possibly have a regulatory role. The analysis of the co-occurrence of human FeS-Ps with other proteins highlighted numerous links between the iron-sulfur cluster machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of reactive oxygen species within the biogenesis and degradation of FeS-Ps.

Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells

Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.

HEAT repeats - versatile arrays of amphiphilic helices working in crowded environments?

Cellular proteins do not work in isolation. Instead, they often function as part of large macromolecular complexes, which are transported and concentrated into specific cellular compartments and function in a highly crowded environment. A central theme of modern cell biology is to understand how such macromolecular complexes are assembled efficiently and find their destinations faithfully. In this Opinion article, we will focus on HEAT repeats, flexible arrays of amphiphilic helices found in many eukaryotic proteins, such as karyopherins and condensins, and discuss how these uniquely designed helical repeats might underlie dynamic protein-protein interactions and support cellular functions in crowded environments. We will make bold speculations on functional similarities between the action of HEAT repeats and intrinsically disordered regions (IDRs) in macromolecular phase separation. Potential contributions of HEAT-HEAT interactions, as well as cooperation between HEATs and IDRs, to mesoscale organelle assembly will be discussed.

Conditional Deletion of Bmal1 in Ovarian Theca Cells Disrupts Ovulation in Female Mice.

Rhythmic events in female reproductive physiology, including ovulation, are tightly controlled by the circadian timing system. The molecular clock, a feedback loop oscillator of clock gene transcription factors, dictates rhythms of gene expression in the hypothalamo-pituitary-ovarian axis. Circadian disruption due to environmental factors (eg, shift work) or genetic manipulation of the clock has negative impacts on fertility. Although the central pacemaker in the suprachiasmatic nucleus classically regulates the timing of ovulation, we have shown that this rhythm also depends on phasic sensitivity to LH. We hypothesized that this rhythm relies on clock function in a specific cellular compartment of the ovarian follicle. To test this hypothesis we generated mice with deletion of the Bmal1 locus in ovarian granulosa cells (GCs) (Granulosa Cell Bmal1 KO; GCKO) or theca cells (TCs) (Theca Cell Bmal1 KO; TCKO). Reproductive cycles, preovulatory LH secretion, ovarian morphology and behavior were not grossly altered in GCKO or TCKO mice. We detected phasic sensitivity to LH in wild-type littermate control (LC) and GCKO mice but not TCKO mice. This decline in sensitivity to LH is coincident with impaired fertility and altered patterns of LH receptor (Lhcgr) mRNA abundance in the ovary of TCKO mice. These data suggest that the TC is a pacemaker that contributes to the timing and amplitude of ovulation by modulating phasic sensitivity to LH. The TC clock may play a critical role in circadian disruption-mediated reproductive pathology and could be a target for chronobiotic management of infertility due to environmental circadian disruption and/or hormone-dependent reprogramming in women.