Daily fluctuations in pollination effectiveness explain higher efficiency of native over exotic bees in Lepechinia floribunda (Lamiaceae).

BACKGROUND: Despite Stebbins' principle of the most efficient pollinator being proposed decades ago, the most important pollinators are still mainly identified using the frequency of visits to flowers. This shortcoming results in a gap between the characterization of the flower visitors of a plant species and a reliable estimation of the plant fitness consequences of the mutualistic interaction. The performance of a mutualistic visitor depends on its abundance, behaviour, effectiveness (pollen removal and deposition per unit time) and efficiency (seed set per unit time) conditioned by the temporal matching between pollinator activity and temporal patterns of maturation of the sexual functions of flowers. Although there have been recent attempts to provide a conceptual and methodological framework to characterize pollinators' performance, few have combined all key elements of visitors and plants to provide an accurate estimation of pollinators' performance under natural conditions. METHODS: We complement information on the flower biology and mating system of the sub-shrub Lepechinia floribunda (Lamiaceae) to provide a daily quantitative estimation of performance (effectiveness and efficiency) of the more abundant pollinators, i.e. native bumble-bees (Bombus spp.) and leafcutter bees (Megachile sp.), and the exotic honey-bee (Apis mellifera). KEY RESULTS: Unlike honey-bees or leafcutter bees, native bumble-bees matched the daily pattern of nectar production and stigma receptivity, and showed higher effectiveness and efficiency. Despite the overabundance of honey-bees, visits occurred mainly when stigmas were not receptive, thus reducing the honey-bees' overall performance. CONCLUSIONS: Bumble-bees appear to be the most important pollinators and potential historical mediators of reproductive trait evolution in L. floribunda. Because the production of seeds by bumble-bees involved fewer pollen grains for plants and less investment in floral display than honey-bees, contemporary and expected changes in pollinator abundance may affect future L. floribunda floral evolution. If bumble-bees were to be further displaced by anthropogenic disturbance or by competition with honey-bees, their lower efficiency will select for a larger floral display increasing reproductive costs. This scenario may also impose selection to reduce dichogamy to match honey-bee foraging activity.

Short transmembrane domains with high-volume exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex.

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the Golgi SNARE protein Sft1 and the plasma membrane SNARE protein Sso1 from Saccharomyces cerevisiae as model proteins, we modified the length of their TMDs and the volume of their exoplasmic hemi-TMD, and determined their subcellular localization both in yeast and mammalian cells. We found that short TMDs with high-volume exoplasmic hemi-TMDs confer Golgi membrane residence, whereas TMDs with low-volume exoplasmic hemi-TMDs, either short or long, confer plasma membrane residence to these proteins. Results indicate that the shape of the exoplasmic hemi-TMD, in addition to the length of the entire TMD, determine retention in the Golgi or exit to the plasma membrane of Type II membrane proteins.

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Differential endocytic trafficking of neuropathy-associated antibodies to GM1 ganglioside and cholera toxin in epithelial and neural cells.

Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain-Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with co-endocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathy-associated antibodies.

Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway.

GAP-43 (growth-associated protein-43) is a dually palmitoylated protein, at cysteine residues at positions 3 and 4, that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in CHO (Chinese hamster ovary)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and TGN (trans-Golgi network). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of Cys3 and Cys4 of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the plasma membrane pool. Newly synthesized GAP-43 is found mainly associated with the TGN, but not with the pericentriolar recycling endosome, and traffics to the plasma membrane by a brefeldin A-insensitive pathway. Impairment of plasma membrane fusion and internalization by treatment with tannic acid does affect the trafficking of GAP-43 from plasma membrane to recycling endosomes which reveals a vesicle-mediated retrograde trafficking of GAP-43. Here, we also show that internalization of GAP-43 is regulated by Arf (ADP-ribosylation factor) 6. Taken together, these results demonstrate that dual acylation is required for sorting of peripheral membrane-associated GAP-43 to recycling endosome via an Arf6-associated endocytic vesicular pathway.

Retrospective study of the prevalence of human T-cell lymphotropic virus-type 1/2, HIV, and HBV in pregnant women in Argentina.

This study shows first data on HTLV-1/2 seroprevalence among pregnant women in the non-endemic region of Argentina. In a retrospective study a representative sample (n = 3,143) of the pregnant women registered in the health public service in the province of Córdoba was evaluated. HTLV-1/2 seroprevalence was 0.191% +/- 0.0857 [IC 0.022-0.359]. This prevalence was 10 times higher in pregnant women than in blood donors [0.019 (4/21.183)]. The pregnant women would reflect the epidemiology of the general population more accurately since it constitutes a more heterogeneous group than that of blood donors. The prevalence of infection with HIV was 2.8 times higher than that of HTLV-1/2 (P < 0.05) and the presence of any of these two viruses was not a subrogating indicator of the presence of the other (Goodman and Kruskal's Tau coefficient = 0.0092). The prevalence of HBV was not significantly different from that of HTLV-1/2 (P > 0.05). We consider that it is necessary to carry out continuous studies in order to define the main risk factors for infection of these women. Thus, a decision could be made to apply the best policy in public health to prevent vertical transmission of the virus in Argentina.