Phosphatidylserine exposure promotes increased adhesion in Dictyostelium Copine A mutants

The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure.

Morphological characteristics of respiratory and digestive organs of Roman snail (Helix pomatia L., 1758)

The aim of this work was to study the structure of lung and hepatopancreas of Roman snail (Helix of pomatia of L., 1758). The study found that the lung occupies the lower turn of shell and presented by a saccate cavity, in the wall of that there are a kidney and heart with a pericardium, and also a rectum and ureter pass. An external surface of lungs covered by a shell and covered by an epidermis. An internal surface is covered by a flat ciliated epithelium and forms numerous folds in which pulmonary vessels and lacunae are accommodated. The branches of pulmonary vein have a thick muscular wall, that consists of circular and longitudinal muscular layers. An internal surface of lungs covered by the layer of mucus. Inhalation and exhalation are carried out due to reduction and relaxation of muscles of dorsal wall of the body that is named a “diaphragm”. Gas exchange occurs through the hemolymphatic capillaries of the lung wall. Respiratory motions take place not rhythmically, but through the different intervals of time depending on a requirement in oxygen. The frequency of pneumostome closing and opening is typically one time in a minute. At subzero humidity of atmospheric air of pneumostome closed by a mantle, and also one (or a few) epiphragms. The hepatopancreas (“liver” or liver gland) is in the upper rotation of the sink and formed by two parts: right and left, from which two liver ducts enter into the stomach respectively. The liver gland consists of many acinuss, surrounded by connecting tissue, that contains small number of muscular fibres. Calcium cells have a pyramidal form and usually do not reach the lumen of the acinus. Cytoplasm of calcium cells contains inclusions: grains of phosphoricacid lime and drops of fat. The digestive cells of the hepatopencreas are more elongated, often clavicular. Сytoplasm of digestive cells is loose and vacuolated and contain inclusions of yellow-green color. Enzyme cells on histopreparations are difficult to distinguish from digestive ones. They contain transparent vacuoles with a large round inclusion of yellow-green color, which consists of a cluster of several grains of different sizes. Hepatopancreas performs the following functions: secretory (enzyme cells), absorption and intracellular digestion (digestive cells), preservation of nutrients and calcium (calcium cells), and also excretory function.

Distinct pathways for zinc metabolism in the terrestrial slug Arion vulgaris

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.

5. The hard stuff: calcium, cells, bones, and cancer

‘The hard stuff: calcium, cells, bones, and cancer’ considers calcium and the hormone systems that regulate it. These hormones keep the correct balance between the highly reactive Ca2+ ions, which are essential for many life processes, and the precipitating salts like carbonates and phosphates. Controlled precipitation builds bones that defy the pull of gravity and hard materials that protect against predators. Bone formation and recycling take energy, as well as calcium and phosphorus, and all aspects of skeletal development are affected by the hormones of growth and metabolism. There are connections between bone development and appetite which work through serotonin, leptin, insulin, and hormones produced by the gut, in addition to calcitonin.

Adrenergically activated Ca2+ increases in brown fat cells: effects of Ca2+, K+, and K channel block

We measured intracellular calcium concentration ([Ca2+]i) during adrenergic stimulation using fura-2 ratio imaging of individual cultured neonatal rat brown fat cells. One micromolar norepinephrine (NE) increased [Ca2+]i from an average resting value of 105 nM to 555 nM in approximately 30 s. [Ca2+]i remained elevated as long as NE was present but returned to resting levels within 2-3 min after NE removal. The response was half maximal at approximately 50 nM NE and was primarily alpha-adrenergic. The sustained, but not the initial, increase in [Ca2+]i required extracellular calcium. Cells stimulated in high-K media had [Ca2+]i responses like those in 0 Ca2+, suggesting that depolarization abrogates calcium influx. Parallel perforated-patch recordings showed that the increase in [Ca2+]i activates a calcium-activated K conductance. Blocking K channels with moderate concentrations of tetraethylammonium (TEA) had only small effects on NE-induced changes in [Ca2+]i, but high concentrations of TEA significantly reduced the response. We conclude that cytoplasmic calcium is modulated by fluxes from both intracellular and extracellular sources and that K channels may not be required for normal short-term [Ca2+]i responses to hormone.

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl- L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F- actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours of maturation. The PKC inhibitor 1–5- isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F- actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a “mature” response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl- L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F- actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours of maturation. The PKC inhibitor 1–5- isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F- actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a “mature” response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

Corrosion of intracellular granules and cell death

The snail Helix aspersa has large numbers of calcium cells in its hepatopancreas which contain membrane-bound intracellular granules of CaMgP 2 O 7 . These inorganic deposits are the sites of accumulation of a wide variety of cations and act as a detoxification mechanism that traps a number of dietary metals once they have entered the animal. This study concentrates on the mechanism of incorporation of manganese ions into these deposits by using electron microscopy, ultra-low-angle X-ray scattering and extended X-ray absorption spectroscopy. The results show that manganese ions cause a localized corrosion of these intracellular granules with a consequent release of calcium ions. This release appears to overwhelm the calcium-regulatory mechanisms of these cells and leads to their death and subsequent shedding into the lumen of the alimentary tract. The concept of intracellular corrosion is a novel interpretation of a previously unsuspected biochemical lesion.