Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease-activated receptor-2 internalization.

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred-either in a membrane-bound melanosome or as a melanosome core, that is, melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1, CtBP1/BARS, Cdc42 or RhoA, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct routes of melanin internalization. Furthermore, we show that melanocore uptake induces protease-activated receptor-2 (PAR-2) internalization by keratinocytes to a higher extent than melanosomes. Because skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore-based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cell homeostasis.

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, which is characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMC homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam-cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMC proliferation and migration, impacting atherosclerosis. In summary, this work shows that ChA is sufficient to induce lysosomal dysfunction in VSMCs, that, in ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered, and, finally, that recombinant LAL can be a therapeutic approach for lysosomal dysfunction.

Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB.

Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of Ca2+-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) as well as Rab3a, which we have previously described to be a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.

Performance of the ACMG-AMP criteria in a large familial renal glucosuria cohort with identified SLC5A2 sequence variants.

Familial Renal Glucosuria (FRG) is a co-dominantly inherited trait characterized by orthoglycaemic glucosuria. From 2003 to 2015 we have reported several cohorts validating SLC5A2 (16p11.2), encoding SGLT2 (Na+/glucose cotransporter family member 2), as the gene responsible for FRG. The aim of this work was to validate the variants identified in our extended FRG cohort of published, as well more recent unreported cases, according to the ACMG-AMP 2015 criteria. Forty-six variants were evaluated, including 16 novel alleles first described in this study. All are rare, ultra-rare or absent from population databases and most are missense changes. According to the ACMG-AMP standards, only 74% of the variants were classified as P/LP. The lack of descriptions of unrelated patients with similar variants or failing to test additional affected family members, averted a conclusion for pathogenicity in the alleles that scored VUS, highlighting the importance of both family testing and variant reporting. Finally, the cryo-EM structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state improved the ACMG-AMP pathogenicity score by identifying critical/functional protein domains.

Plant Responses to Climate Change.

Ongoing climate change poses a great risk to the natural environment and the sustainability of agriculture [...].

Overexpression of Water-Responsive Genes Promoted by Elevated CO2 Reduces ROS and Enhances Drought Tolerance in Coffea Species.

Drought is a major constraint to plant growth and productivity worldwide and will aggravate as water availability becomes scarcer. Although elevated air [CO2] might mitigate some of these effects in plants, the mechanisms underlying the involved responses are poorly understood in woody economically important crops such as Coffea. This study analyzed transcriptome changes in Coffea canephora cv. CL153 and C. arabica cv. Icatu exposed to moderate (MWD) or severe water deficits (SWD) and grown under ambient (aCO2) or elevated (eCO2) air [CO2]. We found that changes in expression levels and regulatory pathways were barely affected by MWD, while the SWD condition led to a down-regulation of most differentially expressed genes (DEGs). eCO2 attenuated the impacts of drought in the transcripts of both genotypes but mostly in Icatu, in agreement with physiological and metabolic studies. A predominance of protective and reactive oxygen species (ROS)-scavenging-related genes, directly or indirectly associated with ABA signaling pathways, was found in Coffea responses, including genes involved in water deprivation and desiccation, such as protein phosphatases in Icatu, and aspartic proteases and dehydrins in CL153, whose expression was validated by qRT-PCR. The existence of a complex post-transcriptional regulatory mechanism appears to occur in Coffea explaining some apparent discrepancies between transcriptomic, proteomic, and physiological data in these genotypes.

Editorial: Tropical Plant Responses to Climate Change.

The climate crisis is pushing the planet's tropical plants towards their limits [...].

Rab35 controls cilium length, function and membrane composition.

Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left-right organiser (Kupffer's vesicle) and causes motile cilia-associated left-right asymmetry defects. Consistent with these observations, GFP-Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5-phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators.

A Transcriptomic Approach to Understanding the Combined Impacts of Supra-Optimal Temperatures and CO2 Revealed Different Responses in the Polyploid Coffea arabica and Its Diploid Progenitor C. canephora.

Understanding the effect of extreme temperatures and elevated air (CO2) is crucial for mitigating the impacts of the coffee industry. In this work, leaf transcriptomic changes were evaluated in the diploid C. canephora and its polyploid C. arabica, grown at 25 °C and at two supra-optimal temperatures (37 °C, 42 °C), under ambient (aCO2) or elevated air CO2 (eCO2). Both species expressed fewer genes as temperature rose, although a high number of differentially expressed genes (DEGs) were observed, especially at 42 °C. An enrichment analysis revealed that the two species reacted differently to the high temperatures but with an overall up-regulation of the photosynthetic machinery until 37 °C. Although eCO2 helped to release stress, 42 °C had a severe impact on both species. A total of 667 photosynthetic and biochemical related-DEGs were altered with high temperatures and eCO2, which may be used as key probe genes in future studies. This was mostly felt in C. arabica, where genes related to ribulose-bisphosphate carboxylase (RuBisCO) activity, chlorophyll a-b binding, and the reaction centres of photosystems I and II were down-regulated, especially under 42°C, regardless of CO2. Transcriptomic changes showed that both species were strongly affected by the highest temperature, although they can endure higher temperatures (37 °C) than previously assumed.

Transcriptomic Leaf Profiling Reveals Differential Responses of the Two Most Traded Coffee Species to Elevated [CO2].

As atmospheric [CO2] continues to rise to unprecedented levels, understanding its impact on plants is imperative to improve crop performance and sustainability under future climate conditions. In this context, transcriptional changes promoted by elevated CO2 (eCO2) were studied in genotypes from the two major traded coffee species: the allopolyploid Coffea arabica (Icatu) and its diploid parent, C. canephora (CL153). While Icatu expressed more genes than CL153, a higher number of differentially expressed genes were found in CL153 as a response to eCO2. Although many genes were found to be commonly expressed by the two genotypes under eCO2, unique genes and pathways differed between them, with CL153 showing more enriched GO terms and metabolic pathways than Icatu. Divergent functional categories and significantly enriched pathways were found in these genotypes, which altogether supports contrasting responses to eCO2. A considerable number of genes linked to coffee physiological and biochemical responses were found to be affected by eCO2 with the significant upregulation of photosynthetic, antioxidant, and lipidic genes. This supports the absence of photosynthesis down-regulation and, therefore, the maintenance of increased photosynthetic potential promoted by eCO2 in these coffee genotypes.

Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport.

Microtubules and F-actin, and their associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes, myosin-Va and kinesin-1 have both been proposed as long-range centrifugal transporters moving melanosomes into the peripheral dendrites. Here, we investigated the role of kinesin-1 heavy chain (Kif5b) and its suggested targeting factor Rab1a in transport. We performed confocal microscopy and subcellular fractionation, but did not detect Kif5b or Rab1a on melanosomes. Meanwhile functional studies, using siRNA knockdown and dominant negative mutants, did not support a role for Kif5b or Rab1a in melanosome transport. To probe the potential of Kif5b to function in transport, we generated fusion proteins that target active Kif5b to melanosomes and tested their ability to rescue perinuclear clustering in myosin-Va-deficient cells. Expression of these chimeras, but not full-length Kif5b, dispersed melanosomes with similar efficiency to myosin-Va. Our data indicate that kinesin and microtubules can compensate for defects in myosin-Va and actin-based transport in mammals, but that endogenous Kif5b does not have an important role in transport of melanocytes due to its inefficient recruitment to melanosomes.

Actinorhizal trees and shrubs from Africa: distribution, conservation and uses.

Actinorhizal plants are a group of perennial dicotyledonous angiosperms, comprised of more than 200 species, most of which can establish root-nodule symbiosis with the nitrogen fixing actinobacteria of the genus Frankia. They are key providers of fundamental goods and services and can give a major contribution to mitigate the combined effects of climate changes, human population growth and loss of biodiversity. This aspect is particularly relevant for the developing economies of many African countries, which are highly exposed to climate and anthropogenic disturbances. In this work we have analyzed the distribution, conservation and uses of actinorhizal species native to or introduced in Africa. A total of 42 taxa distributed over six botanical families (Betulaceae, Casuarinaceae, Myricaceae, Elaeagnaceae, Rhamnaceae and Coriariaceae) were identified. The vast majority is able to thrive under a range of diverse environments and has multiple ecological and economic potential. More than half of the identified species belong to the genus Morella (Myricaceae), most of them native to Middle, Eastern and Southern Africa. Although the information about the conservation status and uses of Morella spp. is largely incomplete, the available data is indicative of their potential in e.g. forestry and agroforestry, food and medicine. Therefore, efforts should be made to upgrade actinorhizal research in Africa towards the sustainable use of biodiversity at the service of local (bio)economies.

shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity.

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.

Comparative Proteomic Analysis of Nodulated and Non-Nodulated Casuarina glauca Sieb. ex Spreng. Grown under Salinity Conditions Using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS).

Casuarina glauca displays high levels of salt tolerance, but very little is known about how this tree adapts to saline conditions. To understand the molecular basis of C. glauca response to salt stress, we have analyzed the proteome from branchlets of plants nodulated by nitrogen-fixing Frankia Thr bacteria (NOD+) and non-nodulated plants supplied with KNO3 (KNO3+), exposed to 0, 200, 400, and 600 mM NaCl. Proteins were identified by Short Gel, Long Gradient Liquid Chromatography coupled to Tandem Mass Spectrometry and quantified by Sequential Window Acquisition of All Theoretical Mass Spectra -Mass Spectrometry. 600 proteins were identified and 357 quantified. Differentially Expressed Proteins (DEPs) were multifunctional and mainly involved in Carbohydrate Metabolism, Cellular Processes, and Environmental Information Processing. The number of DEPs increased gradually with stress severity: (i) from 7 (200 mM NaCl) to 40 (600 mM NaCl) in KNO3+; and (ii) from 6 (200 mM NaCl) to 23 (600 mM NaCl) in NOD+. Protein-protein interaction analysis identified different interacting proteins involved in general metabolic pathways as well as in the biosynthesis of secondary metabolites with different response networks related to salt stress. Salt tolerance in C. glauca is related to a moderate impact on the photosynthetic machinery (one of the first and most important stress targets) as well as to an enhancement of the antioxidant status that maintains cellular homeostasis.

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors.

Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle.

Heavy metal content of edible plants collected close to an area of intense mining activity (southern Portugal).

The Neves-Corvo mining complex (MC) situated in southern Portugal exploits one of the most world's important copper deposits. Agricultural soils surrounding the MC, used by the inhabitants for crop production, contain excessive amounts of As, Cu, Pb, and Zn. Thus, a potential risk to human consumption exists if edible plants grow on these substrata. Arsenic and Pb were not detected in edible samples collected near the MC and 5 km away, but in the leaves-structural or adsorbed onto the surface. In general, Zn was the most mobile element in both contaminated and reference areas as seen by the bioaccumulation factors (BAF). The tolerable upper intake (TUI) values for Cu are a reason of concern, since in 57.1% of the cases, the TUI values are above the recommended upper limit of 5 mg/day, in the case of Ficus carica, Cucurbita pepo, and Phaseolus vulgaris, whereas in 28.6% of the cases, the TUI values are near this limit (C. pepo and Citrus x sinensis). The consumption of such vegetables from these areas must be banned or strongly reduced, since long-term accumulation of Cu can cause a chronic toxicity in humans.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response.

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose-phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

Host cell autophagy contributes to Plasmodium liver development.

Autophagy plays an important role in the defence against intracellular pathogens. However, some microorganisms can manipulate this host cell pathway to their advantage. In this study, we addressed the role of host cell autophagy during Plasmodium berghei liver infection. We show that vesicles containing the autophagic marker LC3 surround parasites from early time-points after invasion and throughout infection and colocalize with the parasitophorous vacuole membrane. Moreover, we show that the LC3-positive vesicles that surround Plasmodium parasites are amphisomes that converge from the endocytic and autophagic pathways, because they contain markers of both pathways. When the host autophagic pathway was inhibited by silencing several of its key regulators such as LC3, Beclin1, Vps34 or Atg5, we observed a reduction in parasite size. We also found that LC3 surrounds parasites in vivo and that parasite load is diminished in a mouse model deficient for autophagy. Together, these results show the importance of the host autophagic pathway for parasite development during the liver stage of Plasmodium infection.

Host PI(3,5)P2 activity is required for Plasmodium berghei growth during liver stage infection.

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5-kinase (PIKfyve) that converts phosphatidylinositol 3-phosphate [PI(3)P] into phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2 ] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non-pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P2 effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P2 -dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.