A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

Balearic insular isolation and large continental spread framed the phylogeography of the western Mediterranean Cheirolophus intybaceus s.l. (Asteraceae)

Recent Quaternary geological and climate events have shaped the evolutionary histories of plant species in the Mediterranean basin, one of the most important hotspots of biodiversity. Genetic analyses of the western Mediterranean Cheirolophus intybaceus s.l. (Asteraceae) based on AFLP were conducted to establish the relationships between its close species and populations, to reconstruct the phylogeography of the group and to analyse potential unidirectional versus bidirectional dispersals between the Ibero‐Provençal belt and the Balearic Islands. AFLP data revealed two main genetic groups, one constituted by the Balearic populations and Garraf (NE Iberia) and the other formed by the remaining mainland populations that were further sub‐structured into two geographically separated subgroups (SE + E Iberia and NE Iberia + SW France). Genetic diversity and spatial structure analyses suggested a mid‐Pleistocene scenario for the origin of C. intybaceus in southern Iberia, followed by dispersal to the north and a single colonisation event of the Balearic archipelago from the near Dianic NE Iberian area. This hypothesis was supported by paleogeographic data, which showed the existence of terrestrial connections between the continent and the islands during the Middle–Late Pleistocene marine regressions, whereas the more recent single back‐colonisation of the mainland from Mallorca might be explained by several hypotheses, such as long‐distance dispersal mediated by migratory marine birds or sea currents.     

Effect of habitat disturbance on pollination biology of the columnar cactus Stenocereus quevedonis at landscape‐level in central Mexico

Stenocereus quevedonis (‘pitire’) is a columnar cactus endemic to central Mexico, grown for its edible fruit. Phenology, pollination biology and behaviour of flower visitors of this species were compared in six conserved and disturbed sites, hypothesising that: (i) pitire pollination is self‐incompatible, requiring animal vectors; (ii) higher incidence of radiation on plants in cleared forest may lead to a higher number of flowers per pitire plant and longer blooming season, and disturbing and differential spatial availability of flower resources may determine differential attraction of pollinators to conserved and disturbed areas; (iii) if pitire pollination system is specialised, reproductive success would decrease with pollinator scarcity, or other species may substitute for main pollinators. In all sites, pitire reproduction started in January, flowering peak occurring in April, anthesis duration was 15 h and predominantly nocturnal (9 h), pollen was released at 23:00 h, nectar was produced throughout anthesis, and breeding system was self‐incompatible. Flower production per plant was similar in disturbed and conserved sites, but flower availability was higher (because of higher tree density) and longer in disturbed sites. Pollination is nocturnal, the most frequent legitimate pollinator being the bat Leptonycteris yerbabuenae; diurnal pollination is rare but possible, carried out by bee species. Fruit and seed set in control and nocturnal pollination treatments at disturbed sites were higher than in conserved sites. Frequency of L. yerbabuenae visits was similar among site types, but more visits of complementary nocturnal and diurnal pollinators were recorded in disturbed sites, which could explain differences in reproductive success.     

A pilot project testing the effectiveness of three weed control methods on the removal of Lantana camara in Forty Mile Scrub National Park,Queensland, Australia

Lantana (Lantana camara) poses a serious threat to the biodiversity of the dry rainforest vegetation at Forty Mile Scrub National Park, Queensland, Australia, by outcompeting native species and increasing vulnerability to fire. This pilot study tests the effectiveness of three weed control methods (hand pulling, a glyphosphate‐based foliar spray herbicide and a picloram‐ and triclopyr‐based basal bark herbicide) in removing lantana and their success in reducing lantana fuel loads. The foliar spray herbicide was the most effective in killing lantana, while manual pulling resulted in the largest decrease in fuel height. We suggest that foliar spraying will be most efficient for combating large infestations of lantana, while hand pulling techniques are recommended for creating firebreaks or when minimizing damage to native species is paramount.     

Provisioning habitat with custom‐designed nest‐boxes increases reproductive success in an endangered finch

Manipulating resource availability to assess the strength and effects of resource limitation on an animal population is relatively straightforward, and thus, common in the scientific literature. Resource quality, however, is rarely manipulated, as this requires a priori knowledge of a relationship between some measurable variation in the resource and individual preference and/or fitness. Recent research on nest‐site selection was used to inform the design of custom‐built nest‐boxes for an endangered hollow‐nester, the Gouldian finch (Erythrura gouldiae). By provisioning breeding populations with these ‘high quality’ artificial nest sites over 3 years, we experimentally investigated the strength and effects of nest‐site limitation in this species. Breeding pairs using nest‐boxes initiated nesting earlier, produced larger clutches and fledged more offspring per season than those in natural hollows. Total and mean reproductive output also increased in nest‐box provisioned sites over the duration of the study. All of these effects were predominantly driven by pairs initiating clutches earlier, potentially because of the reduced intra‐ and interspecific competition for nest sites at optimal breeding times. Our findings suggest that reproduction in wild Gouldian finch populations may be limited by the availability of high quality nest sites in the landscape, and that nest‐boxes could be used as a tool for enhancing reproduction in recovering populations. Furthermore, we conclude that resource quality is equally, if not more important than quantity when supplementing resource‐limited habitats.     

Rainfall in prior breeding seasons influences population size of a small marsupial

The imposition of the stresses of climate change (higher temperatures and in many regions lower rainfall) on existing stressors, such as habitat loss and degradation, will increase pressures on native fauna already experiencing declines. We focused on assessing how the ‘Big Dry’ (severe drought, 1997–2010) in south‐eastern Australia affected populations of a small marsupial carnivore, the yellow‐footed antechinus (Antechinus flavipes), in box‐ironbark forests, which suffer a range of anthropogenic disturbances. Trapping of the mammal was conducted on 136 (0.25 ha) sites in two box‐ironbark forests in 2004, 2005 and 2011 (46 or 64 sites per year). Capture rates of all distinct individuals, males and second‐year females with suckled teats, and the number of suckled teats were positively associated with rainfall in the previous September (time of lactation and deposition of young in nests). Despite differences between forests in capture rates of all individuals, the positive effect of rainfall was evident in both forests. Populations in one forest, Chiltern, were substantially larger than other locations surveyed in 2004 and 2005, yet crashed to small numbers in 2011. This crash was most likely due to low rainfall in the preceding years including the lowest recorded annual rainfall (2006), below‐average annual rainfall (2007, 2008 and 2009) and well‐below‐average rainfall in September (2006, 2007 and 2008). The predicted drying and warming climate in south‐eastern Australia and habitat loss and degradation pose a threat to the viability of the yellow‐footed antechinus in box‐ironbark forests. An integrated approach to small‐mammal management is necessary given that the region may be facing additional losses, especially during droughts, to those already experienced since the early 1800s. Our work emphasizes the need to identify specific effects of stressors on vital demographic characteristics of species.