Spatial insurance against a heatwave differs between trophic levels in experimental aquatic communities

Climate change-related heatwaves are major threats to biodiversity and ecosystem functioning. However, our current understanding of the mechanisms governing community resistance to and recovery from extreme temperature events is still rudimentary. The spatial insurance hypothesis postulates that diverse regional species pools can buffer ecosystem functioning against local disturbances through the immigration of better-adapted taxa. Yet, experimental evidence for such predictions from multi-trophic communities and pulse-type disturbances, like heatwaves, is largely missing. We performed an experimental mesocosm study to test whether species dispersal from natural lakes prior to a simulated heatwave could increase the resistance and recovery of plankton communities. As the buffering effect of dispersal may differ among trophic groups, we independently manipulated the dispersal of organisms from lower (phytoplankton) and higher (zooplankton) trophic levels. The experimental heatwave suppressed total community biomass by having a strong negative effect on zooplankton biomass, probably due to a heat-induced increase in metabolic costs, resulting in weaker top-down control on phytoplankton. While zooplankton dispersal did not alleviate the negative heatwave effects on zooplankton biomass, phytoplankton dispersal enhanced biomass recovery at the level of primary producers, providing partial evidence for spatial insurance. The differential responses to dispersal may be linked to the much larger regional species pool of phytoplankton than of zooplankton. Our results suggest high recovery capacity of community biomass independent of dispersal. However, community composition and trophic structure remained altered due to the heatwave, implying longer-lasting changes in ecosystem functioning.     

Affinity of fibroblast growth factors for beta-cyclodextrin tetradecasulfate

beta-Cyclodextrin tetradecasulfate was found to have a very strong affinity for fibroblast growth factor (FGF) and could substitute for heparin in FGF purification. Basic FGF was purified about 200,000-fold from a rat chondrosarcoma using a method of biaffinity chromatography in which the beta-cyclodextrin tetradecasulfate polymer was mixed with copper-Sepharose. This method takes advantage of the strong affinity of FGF for both beta-cyclodextrin tetradecasulfate and copper.     

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.     

Long-term bovine hematopoietic engraftment with clone-derived stem cells

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.     

Specific N-glycans direct apical delivery of transmembrane, but not soluble or glycosylphosphatidylinositol-anchored forms of endolyn in Madin-Darby canine kidney cells

The sialomucin endolyn is a transmembrane protein with a unique trafficking pattern in polarized Madin-Darby canine kidney cells. Despite the presence of a cytoplasmic tyrosine motif that, in isolation, is sufficient to mediate basolateral sorting of a reporter protein, endolyn predominantly traverses the apical surface en route to lysosomes. Apical delivery of endolyn is disrupted in tunicamycin-treated cells, implicating a role for N-glycosylation in apical sorting. Site-directed mutagenesis of endolyn's eight N-glycosylation sites was used to identify two N-glycans that seem to be the major determinants for efficient apical sorting of the protein. In addition, apical delivery of endolyn was disrupted when terminal processing of N-glycans was blocked using glycosidase inhibitors. Missorting of endolyn occurred independently of the presence or absence of the basolateral sorting signal, because apical delivery was also inhibited by tunicamycin when the cytoplasmic tyrosine motif was mutated. However, we found that apical secretion of a soluble mutant of endolyn was N-glycan independent, as was delivery of glycosylphosphatidylinositol-anchored endolyn. Thus, specific N-glycans are only essential for the apical sorting of transmembrane endolyn, suggesting fundamental differences in the mechanisms by which soluble, glycosylphosphatidylinositol-anchored, and transmembrane proteins are sorted.     

Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the mu2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an approximately 300-residue region of AIP1/Alix-(409-715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 mu2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant mu2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities.     

Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells

Channel gating ofthe cystic fibrosis transmembrane conductance regulator (CFTR) isactivated in response to cAMP stimulation. In addition, CFTR activationmay also involve rapid insertion of a subapical pool of CFTR into theplasma membrane (PM). However, this issue has been controversial, inpart because of the difficulty in distinguishing cell surface vs.intracellular CFTR. Recently, a fully functional, epitope-tagged formof CFTR (M2-901/CFTR) that can be detected immunologically innonpermeabilized cells was characterized (Howard M, Duvall MD,Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J PhysiolCell Physiol 269: C1565-C1576, 1995; and Schultz BD,Takahashi A, Liu C, Frizzell RA, and Howard M. Am J PhysiolCell Physiol 273: C2080-C2089, 1997). We have developedreplication-defective recombinant adenoviruses that expressM2-901/CFTR and used them to probe cell surface CFTR in forskolin(FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells.Virally expressed M2-901/CFTR was functional and was readilydetected on the apical surface of FSK-stimulated polarized MDCK cells.Interestingly, at low multiplicity of infection, we observedFSK-stimulated insertion of M2901/CFTR into the apical PM, whereas athigher M2-901/CFTR expression levels, no increase in surfaceexpression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSKstimulation and demonstrates that the apically insertedM2-901/CFTR originates from a population of subapical vesicles.Our observations may reconcile previous conflicting reports regardingthe effect of cAMP stimulation on CFTR trafficking.

     

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.