Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

Super-resolution microscopy: a comparative treatment.

One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.

HTS-Compatible Patient-Derived Cell-Based Assay to Identify Small Molecule Modulators of Aberrant Splicing in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3'UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG(5)) and DM1 patient (DMPK CTG(1000)) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.

Filopodia are induced by aquaporin-9 expression.

Understanding filopodial formation in motile cells is a pertinent task in cell biology. In the present study we show that expression of the human water channel aquaporin-9 (AQP9) in different cell lines induces the formation of numerous filopodial extensions. Several lines of evidence support the role of aquaporins functioning both as a water channel and signaling participant. The number of filopodia is decreased by site-directed serine substitutions in putative PKC-binding or -phosphorylation sites at amino acid position 11 and 222 in AQP9. The filopodial phenotype obtained with wild-type AQP9 is associated with elevated levels of active Cdc42, while serine-deleted mutants have reduced levels of GTP-Cdc42. Co-transfection with inhibitory N-WASP CRIB completely abolishes wild-type AQP9-induced filopodia formation. Active PKC(zeta) phosphorylates wild-type AQP9 and myristoylated PKC(zeta) pseudosubstrate inhibits the formation of filopodia in AQP9-expressing cells. Expression of wild-type AQP9, but not mock or serine substituted mutants, increases sensitivity to hypo-osmolaric conditions, yielding a rapid morphological rounding of cells and cell death starting as early as 24 h post-transfection. We propose that increased water influx through AQP9 is critically involved in the formation of membrane protrusions, and that AQP9-induced actin polymerization is augmented by activation of Cdc42 and PKC(zeta).

Cdc42 and ARP2/3-independent regulation of filopodia by an integral membrane lipid-phosphatase-related protein.

Filopodia are dynamic cell surface protrusions that are required for proper cellular development and function. We report that the integral membrane protein lipid-phosphatase-related protein 1 (LPR1) localizes to and promotes the formation of actin-rich, dynamic filopodia, both along the cell periphery and the dorsal cell surface. Regulation of filopodia by LPR1 was not mediated by cdc42 or Rif, and is independent of the Arp2/3 complex. We found that LPR1 can induce filopodia formation in the absence of the Ena/Vasp family of proteins, suggesting that these molecules are not essential for the development of the protrusions. Mutagenesis experiments identified residues and regions of LPR1 that are important for the induction of filopodia. RNA interference experiments in an ovarian epithelial cancer cell line demonstrated a role for LPR1 in the maintenance of filopodia-like membrane protrusions. These observations, and our finding that LPR1 is a not an active lipid phosphatase, suggest that LPR1 may be a novel integral membrane protein link between the actin core and the surrounding lipid layer of a nascent filopodium.

Enzymatic analysis of lipid phosphate phosphatases.

Lipid phosphate monoesters including phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate are intermediates in phosho- and sphingo-lipid biosynthesis and also play important roles in intra- and extra-cellular signaling. Dephosphorylation of these lipids terminates their signaling actions and, in some cases, generates products with additional biological activities or metabolic fates. The key enzymes responsible for dephosphorylation of these lipid phosphate substrates are collectively termed lipid phosphate phosphatases (LPPs). They are integral membrane enzymes with a core domain of six transmembrane spanning alpha-helices linked by extramembrane loops. LPPs are oriented in the membrane with their N- and C-termini facing the cytoplasm. LPPs exhibit isoform and cell specific localization patterns being variably distributed between endomembrane compartments (primarily the endoplasmic reticulum and Golgi apparatus) and the plasma membrane. The active site of these enzymes is formed from residues within two of the extramembrane loops and faces the lumen of endomembrane compartments or, when localized to the plasma membrane, towards, the extracellular space. Biochemical, pharmacological, cell biological and genetic studies identify roles for LPPs in both intracellular lipid metabolism and the regulation of both intra- and extra-cellular signaling pathways that control cell growth, survival and migration. This article describes procedures for the expression of LPPs in insect and mammalian cells and their analysis by SDS-PAGE and Western blotting. The most straightforward way to determine LPP activity is to measure release of the substrate phosphate group. We described methods for the synthesis and purification of [(32)P]-labeled LPP substrates. We describe the use of both radiolabeled and fluorescent lipid substrates for the detection, quantitation and analysis of the enzymatic activities of the LPPs measured using intact or broken cell preparations as the source of enzyme.

Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions.

Phospholipids and sphingolipids play critical roles in signal transduction, intracellular membrane trafficking, and control of cell growth and survival. We discuss recent progress in the identification and characterization of a family of integral membrane proteins with central roles in bioactive lipid metabolism and signalling. These five groups of homologous proteins, which we collectively term LPTs (lipid phosphatases/phosphotransferases), are characterized by a core domain containing six transmembrane-spanning alpha-helices connected by extramembrane loops, two of which interact to form the catalytic site. LPT family members are localized to all major membrane compartments of the cell. The transmembrane topology of these proteins places their active site facing the lumen of endomembrane compartments or the extracellular face of the plasma membrane. Sequence conservation between the active site of the LPPs (lipid phosphate phosphatases), SPPs (sphingosine phosphate phosphatases) and the recently identified SMSs (sphingomyelin synthases) with vanadium-dependent fungal oxidases provides a framework for understanding their common catalytic mechanism. LPPs hydrolyse LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and structurally-related substrates. Although LPPs can dephosphorylate intracellularly generated substrates to control intracellular lipid metabolism and signalling, their best understood function is to regulate cell surface receptor-mediated signalling by LPA and S1P by inactivating these lipids at the plasma membrane or in the extracellular space. SPPs are intracellularly localized S1P-selective phosphatases, with key roles in the pathways of sphingolipid metabolism linked to control of cell growth and survival. The SMS enzymes catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and diacylglycerol, suggesting a pivotal role in both housekeeping lipid synthesis and regulation of bioactive lipid mediators. The remaining members of the LPT family, the LPR/PRGs (lipid phosphatase-related proteins/plasticity-related genes) and CSS2s (type 2 candidate sphingomyelin synthases), are presently much less well studied. These two groups include proteins that lack critical amino acids within the catalytic site, and could therefore not use the conserved LPT reaction mechanism to catalyse lipid phosphatase or phosphotransferase reactions. In this review, we discuss recent ideas about their possible biological activities and functions, which appear to involve regulation of cellular morphology and, possibly, lipid metabolism and signalling in the nuclear envelope.

Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity.

Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.

The human lipid phosphate phosphatase-3 decreases the growth, survival, and tumorigenesis of ovarian cancer cells: validation of the lysophosphatidic acid signaling cascade as a target for therapy in ovarian cancer.

Lysophosphatidic acid (LPA) is present at elevated concentrations in the ascites and plasma of ovarian cancer patients. Ovarian cancer cells produce and release LPA both constitutively and after stimulation. LPA can induce proliferation, survival, invasiveness, and resistance to chemotherapy of ovarian cancer cells. This suggests that LPA may be critically important for the development or progression of ovarian cancer and is thus a potential target for therapy. In this study, we demonstrate that introduction of the integral membrane protein, human lipid phosphate phosphohydrolase-3 (hLPP-3) enzyme, which hydrolyzes phosphatidic acid, LPA, sphingosine, and ceramide phosphate in vitro with selectivity for LPA, into SKOV3 and OVCAR-3 ovarian cancer cells decreases colony-forming activity, increases apoptosis, and decreases tumor growth in vitro and in vivo. Strikingly, coculture of hLPP-3-expressing cells with nontransfected parental cells decreased the colony-forming activity of the parental cells, compatible with hLPP-3 decreasing levels of an extracellular mediator, likely LPA. Compatible with this contention, the expression of hLPP-3 was associated with increased rates of extracellular LPA hydrolysis. The effects of hLPP-3 on colony-forming activity were substantially reversed by the LPP-resistant LPA analogue, O-methylphosphothionate. The ability of O-methylphosphothionate to ameliorate the effects of hLPP-3, combined with the inability of an enzymatically inactive hLPP-3 to alter cellular function, suggests that the major effect of hLPP-3 was to increase the hydrolysis of extracellular LPA. Thus genetic or pharmacological manipulation of LPA metabolism, receptor activation, or downstream signaling is an attractive approach for therapy of ovarian cancer.