Spatial analysis of 3' phosphoinositide signaling in living fibroblasts, III: influence of cell morphology and morphological Polarity

Activation of phosphoinositide (PI) 3-kinase is a required signaling pathway in fibroblast migration directed by platelet-derived growth factor. The pattern of 3' PI lipids in the plasma membrane, integrating local PI 3-kinase activity as well as 3' PI diffusion and turnover, influences the spatiotemporal regulation of the cytoskeleton. In fibroblasts stimulated uniformly with platelet-derived growth factor, visualized using total internal reflection fluorescence microscopy, we consistently observed localized regions with significantly higher or lower 3' PI levels than adjacent regions (hot and cold spots, respectively). A typical cell contained multiple hot spots, coinciding with apparent leading edge structures, and at most one cold spot at the rear. Using a framework for finite-element modeling with actual cell contact area geometries, we find that although the 3' PI pattern is affected by irregular contact area shape, cell morphology alone cannot explain the presence of hot or cold spots. Our results and analysis instead suggest that these regions reflect different local 3' PI dynamics, specifically through a combination of mechanisms: enhanced PI 3-kinase activity, reduced 3' PI turnover, and possibly slow/constrained 3' PI diffusion. The morphological polarity of the cell may thus bias 3' PI signaling to promote persistent migration in fibroblasts.     

Host range and variability of calcium binding by surface loops in the capsids of canine and feline parvoviruses

Canine parvovirus (CPV) emerged in 1978 as a host range variant of feline panleukopenia virus (FPV). This change of host was mediated by the mutation of five residues on the surface of the capsid. CPV and FPV enter cells by endocytosis and can be taken up by many non-permissive cell lines, showing that their host range and tissue specificity are largely determined by events occurring after cell entry.We have determined the structures of a variety of strains of CPV and FPV at various pH values and in the presence or absence of Ca(2+). The largest structural difference was found to occur in a flexible surface loop, consisting of residues 359 to 375 of the capsid protein. This loop binds a divalent calcium ion in FPV and is adjacent to a double Ca(2+)-binding site, both in CPV and FPV. Residues within the loop and those associated with the double Ca(2+)-binding site were found to be essential for virus infectivity. The residues involved in the double Ca(2+)-binding site are conserved only in FPV and CPV.Our results show that the loop conformation and the associated Ca(2+)-binding are influenced by the Ca(2+) concentration, as well as pH. These changes are correlated with the ability of the virus to hemagglutinate erythrocytes. The co-localization of hemagglutinating activity and host range determinants on the virus surface implies that these properties may be functionally linked. We speculate that the flexible loop and surrounding regions are involved in binding an as yet unidentified host molecule and that this interaction influences host range.     

Spatial Relationships Between an Introduced Snapper and Native Goatfishes on Hawaiian Reefs*

It has been suggested that the introduced blueline snapper (Lutjanus kasmira, Family: Lutjanidae) may adversely affect populations of native fishery species in Hawai’i through competition for spatial or dietary resources, or through predation on young fish. We studied the habitat use patterns of L. kasmira and several native reef fish species using direct observation by SCUBA divers. Habitat use patterns of the yellowtail goatfish (Mulloidichthys vanicolensis, Family: Mullidae) were most similar to those of L. kasmira. Both species were primarily found low in the water column and were closely associated with areas of vertical relief. Individual M. vanicolensis were found higher in the water column when L. kasmirawere present, but L. kasmira were not similarly affected by M. vanicolensis. This finding suggests asymmetrical competition for shelter, in which the dominant L. kasmira displaces M. vanicolensis farther into the water column. This displacement from the protection of the reef could increase the vulnerability of M. vanicolensisto predators and fishers. *The US Government’s right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

FORAGING OF JUVENILE MONK SEALS AT FRENCH FRIGATE SHOALS, HAWAII

Emaciation and poor survivorship of juvenile Hawaiian monk seals at French Frigate Shoals atoll prompted a study of their foraging, using video camera technology ( crittercam ). Nine juveniles between the ages of 1 and 3 yr (six males, three females) were fitted with crittercam to identify their foraging habitat and feeding behavior. All feeding was directed at small (≤ 10 cm), cryptic, benthic prey. Older seals (ages 2 and 3), varied in their foraging intensity with most of their attention directed at shallow atoll depths (10–30 m). In contrast, the three yearlings focused all their feeding in the sand fields (50–100 m) on the atoll's outer slope. Bottom trawls were used to assess the prey abundance of the sand habitat and found 70% of the numerical catch was flounder ( Bothidae ). Extrapolating the yearlings' prey capture rate (0.13/min, derived from the crittercam video) over their total bottom time yielded an estimated 1–1.3 kg/day of flounder. The mean size of flounder (5 ± 1.7 cm) caught in the bottom trawls was close to the size at which larval flounder settle from the plankton (3 cm), suggesting that localized changes in oceanography could directly impact the seals' prey supply. Extensive use of sand communities by young seals may be the strongest link yet identified between juvenile survivorship and oceanographic dynamics.     

Capacitation of bovine spermatozoa by oviduct fluid

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.     

Functions of the SLC36 transporter Pathetic in growth control

Neurons exhibit extreme diversity in size, but whether large neurons have specialized mechanisms to support their growth is largely unknown. Recently, we identified the SLC36 transporter Pathetic (Path) as a factor required for extreme dendrite growth in neurons. Path is broadly expressed, but only neurons with large dendrite arbors or small neurons that are forced to grow large require path for their growth. To gain insight into the basis of growth control by path, we generated additional alleles of path and further examined the apparent specificity of growth defects in path mutants. Here, we confirm our prior finding that loss of path function imposes an upper limit on neuron growth, and additionally report that path likely limits overall neurite length rather than dendrite length alone. Using a GFP knock-in allele of path, we identify additional tissues where path likely functions in nutrient sensing and possibly growth control. Finally, we demonstrate that path regulates translational capacity in a cell type that does not normally require path for growth, suggesting that path may confer robustness on growth programs by buffering translational output. Altogether, these studies suggest that Path is a nutrient sensor with widespread function in Drosophila.     

Cellular Mechanisms Controlling Caspase Activation and Function

Caspases are the primary drivers of apoptotic cell death, cleaving cellular proteins that are critical for dismantling the dying cell. Initially translated as inactive zymogenic precursors, caspases are activated in response to a variety of cell death stimuli. In addition to factors required for their direct activation (e.g., dimerizing adaptor proteins in the case of initiator caspases that lie at the apex of apoptotic signaling cascades), caspases are regulated by a variety of cellular factors in a myriad of physiological and pathological settings. For example, caspases may be modified posttranslationally (e.g., by phosphorylation or ubiquitylation) or through interaction of modulatory factors with either the zymogenic or active form of a caspase, altering its activation and/or activity. These regulatory events may inhibit or enhance enzymatic activity or may affect activity toward particular cellular substrates. Finally, there is emerging literature to suggest that caspases can participate in a variety of cellular processes unrelated to apoptotic cell death. In these settings, it is particularly important that caspases are maintained under stringent control to avoid inadvertent cell death. It is likely that continued examination of these processes will reveal new mechanisms of caspase regulation with implications well beyond control of apoptotic cell death.Apoptosis is a form of programmed cell death that eliminates individual cells within an organism while preserving the overall structure of surrounding tissue. Many of the prominent morphological features of apoptosis were first described in 1972 by Kerr, Wyllie, and Currie (Kerr et al. 1972). However, it was not until the mid-1990s that apoptosis was linked to the activation of the cysteine-dependent aspartate-driven proteases (caspases), which cleave key intracellular substrates to promote cell death (Cerretti et al. 1992; Nicholson et al. 1995; Alnemri et al. 1996; Liu et al. 1996; Thornberry and Lazebnik 1998). Given the critical role that caspases play in dismantling the cell during apoptosis, their activation and subsequent activity are highly regulated. Failure of a cell to properly modulate caspase activity can cause aberrant or untimely apoptotic cell death, potentially leading to carcinogenesis, autoimmunity, neurodegeneration, and immunodeficiency (Thompson 1995; Hanahan and Weinberg 2000; Yuan and Yankner 2000; Li and Yuan 2008).Caspases are synthesized within the cell as inactive zymogens that lack significant protease activity. Thus, caspases are, in essence, regulated from the moment of protein synthesis in that they are not activated until receipt of specific death stimuli (Earnshaw et al. 1999). The primary structure of a caspase is an amino-terminal prodomain and a carboxy-terminal protease domain, which contains the key catalytic cysteine residue. Caspases are categorized as initiator or effector caspases, based on their position in apoptotic signaling cascades. The initiator caspases (caspase-2, -8, -9, and -10) act apically in cell death pathways and all share long, structurally similar prodomains. This group of enzymes is activated through “induced proximity” when adaptor proteins interact with the prodomains and promote caspase dimerization (Boatright et al. 2003; Baliga et al. 2004; Pop et al. 2006; Riedl and Salvesen 2007; Wachmann et al. 2010). In contrast, the effector caspases (caspase-3, -6, and -7) have shorter prodomains and exist in the cell as preformed, but inactive, homodimers. Following cleavage mediated by an initiator caspase, effector caspases act directly on specific cellular substrates to dismantle the cell. Although many individual caspase substrates have been implicated in specific aspects of cellular destruction (e.g., lamin cleavage is required for the efficient packaging of nuclei into small membrane-bound vesicles), recent proteomic approaches have greatly expanded the known repertoire of proteolytic products generated during apoptosis (Van Damme et al. 2005; Dix et al. 2008; Mahrus et al. 2008). Further work will be needed to confirm these findings and to determine how (or if) all of these substrates participate in the apoptotic process (see Poreba et al. 2013), especially as new details emerge on the relationship between posttranslational modifications, like phosphorylation, and caspase cleavage (Dix et al. 2012).     

Evaluation of 2′-α-fluorine modified nucleoside phosphonates as potential inhibitors of HCV polymerase

Ribonucleoside phosphonate analogues containing 2′-α-fluoro modifications were synthesized and their potency evaluated against HCV RNA polymerase. The diphosphophosphonate (triphosphate equivalent) adenine and cytidine analogues displayed potent inhibition of the HCV polymerase in the range of 1.9–2.1 μM, but only modest cell-based activity in the HCV replicon. Pro-drugs of the parent nucleoside phosphonates improved the cell-based activity.     

Characterization of a second vaccinia virus mRNA-decapping enzyme conserved in poxviruses

Vaccinia virus (VACV) encodes enzymes that cap the 5′ end of viral mRNAs, which enhances their stability and translation. Nevertheless, recent studies demonstrated that the VACV D10 protein (VACV-WR_115) decaps mRNA, an enzymatic activity not previously shown to be encoded by a virus. The decapping activity of D10 is dependent on a Nudix hydrolase motif that is also present in the VACV D9 protein (VACV-WR_114), which shares 25% sequence identity with D10. Here, we showed that a purified recombinant VACV D9 fusion protein also decaps mRNA and that this activity was abolished by point mutations in the Nudix hydrolase motif. Decapping was specific for a methylated cap attached to RNA and resulted in the liberation of m⁷GDP. D9 differed from D10 in requiring a longer capped RNA substrate for optimal activity, having greater sensitivity to inhibition by uncapped RNA, and having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA. Since D9 is expressed early in infection and D10 late, we suggest that the two proteins enhance mRNA turnover and manipulate gene expression in a complementary and overlapping manner.