PLEKHM1: Adapting to life at the lysosome

The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.PLEKHM1 (pleckstrin homology domain containing, family M [with RUN domain] member 1) is a ubiquitously expressed protein involved in the regulation of osteoclast function and bone resorption. Recently, it was also described in the context of negatively regulating the endocytic pathway but not autophagy. However, in our recent studies, we show that PLEKHM1 positively regulates the terminal stages of both endocytic and autophagy pathways through direct interaction between PLEKHM1, RAB7, the HOPS complex, and mammalian Atg8 proteins (Fig. 1A). In addition, the PLEKHM1-RAB7-HOPS complex is a direct target for the Salmonella (Salmonella enterica Typhimurium) effector protein SifA (Salmonella-induced filament protein A) that together regulate the Salmonella-containing vacuole (Fig. 1B). Open in a separate windowFigure 1.Model of PLEKHM1 function in the endocytic and autophagic pathways. (A) Domain structure of PLEKHM1 and their interactions. RUN (RUNDC3A/RPIP8, UNC-14 and RUSC1/NESCA); PH1 and PH2 (Pleckstrin homology domain 1 and 2); C1/Zinc finger (C1); HOPS (homotypic fusion and protein sorting). (B) Proposed positioning of PLEKHM1 and its associated complexes in the autophagy and endocytic pathway. PLEKHM1 localizes to late endosomes and lysosomes in an RAB7-dependent manner. The interaction between PLEKHM1, RAB7, and HOPS on vesicles positions these vesicles for tethering and fusion with autophagosomes, through direct interaction with MAP1LC3/GABARAP proteins. The autophagosomes may also fuse with late endosomes/MVBs (multivesicular bodies) in a PLEKHM1-RAB7-HOPS-dependent manner to produce amphisomes, prior to fusion with the lysosome. PLEKHM1-RAB7-HOPS can also be subverted by the Salmonella effector SifA, for the proper maintenance of the Salmonella-containing vacuole (SCV) and Sif (Salmonella-induced filament) formation. mAtg8s, MAP1LC3/GABARAP proteins.Using a 2-pronged approach, we identified PLEKHM1 as an interaction partner of RAB7 in its GTP-bound active state, RAB7(GTP), and MAP1LC3/GABARAP proteins. PLEKHM1 interacts directly with all MAP1LC3/GABARAP proteins through a highly conserved LC3-interaction motif (LIR) located between the Pleckstrin homology domain 1 (PH1) and PH2 domains of PLEKHM1 (Fig. 1A). Endogenous PLEKHM1 colocalizes with LAMP1 at the cytosolic-facing membrane, but not the lumenal side, of LC3B-containing amphisomes/autolysosomes, indicating that PLEKHM1 is an autophagy adaptor protein rather than a selective cargo receptor.Using SILAC (stable isotope labeling of cells in culture)-labeled inducible PLEKHM1 cells, we identified the HOPS complex as a significant interaction partner. The hexameric HOPS complex is an essential component of the late endocytic fusion machinery and is required for autolysosome formation. PLEKHM1 interacts directly with the HOPS complex, mediated by the RUN domain of PLEKHM1 and the C terminus of VPS39 (Fig. 1A) Crucially, PLEKHM1 forms an endogenous complex with HOPS. In the context of vesicle fusion, the HOPS complex acts as a tether to anchor and position the vesicles prior to fusion that is driven by SNARE proteins. Multiple SNARE proteins, such as VAMP7, VAMP8, VTI1B, SNAP29, and STX17 have been described to be required for autophagosome-lysosome fusion. Upon autophagy induction, enhanced PLEKHM1 coprecipitation is detected with the HOPS complex and the autophagosome specific SNARE STX17, reinforcing a role for PLEKHM1 in autophagosome-lysosome fusion.Both RAB7 and the HOPS complex are integral components of the endocytic pathway and, as such, we wanted to test the effect of PLEKHM1 loss on EGFR (epidermal growth factor receptor) degradation. We used 2 epithelial cell lines, HeLa and Hke3. In both instances, loss of PLEKHM1 causes a marked decrease in the rate of EGFR degradation and increases retention in early endosomes. This is in stark contrast to previous reports that used A549 cells and showed that a lack of PLEKHM1 accelerates EGFR degradation. Clearly, cell lines and their background mutations will have to be considered for future studies.In addition to the endocytic pathway, RAB7 and the HOPS complex are essential for the autophagosome-to-autolysosome transition. Therefore, we also wanted to explore this facet of PLEKHM1 function. We generated Plekhm1 KO MEFs to analyze the effects of autophagy flux in the absence of PLEKHM1. Plekhm1 KO MEFs show a block in autophagy, with the accumulation of SQSTM1/p62 and LC3B-II and, using tandem-fluorescence-LC3B as a marker, a decrease in autolysosome formation. Taken together, these findings suggest that PLEKHM1 functions at the point of autophagosome-lysosome fusion (Fig. 1B).Finally, we were interested in testing the functional role of PLEKHM1, and in particular the LIR, during selective autophagy of protein aggregates. We treated control and PLEKHM1-depleted cells with puromycin and observed aggregate clearance over time after puromycin removal. Cells lacking PLEKHM1 and those reconstituted with a PLEKHM1-LIR mutant were unable to efficiently remove SQSTM1-ubiquitin-positive aggregates, compared to control or PLEKHM1-wild type reconstituted cells, indicating an important role for the final stages of endosome and autophagosome maturation (Fig. 1B).“No man is an island, entire of itself” seems of particular prudence when considering the intertwined nature of both autophagic and endocytic pathways. Indeed, it is interesting that there are multiple RAB7 effector proteins functioning at the late endocytic step that also contribute to autophagy, including FYCO1, KIAA0226/Rubicon, UVRAG and now PLEKHM1, where only PLEKHM1 and UVRAG have been shown to interact with the HOPS complex. All of which, when mutated or depleted, have effects on both the endocytic and autophagic pathways. Clearly the roles of these proteins in cell-type and tissue-specific settings have to be determined before we fully comprehend the complexities of how the endosomal and autophagic pathways integrate and communicate with each other.     

Revisiting the Freedom of Fishing and Legal Obligations on States Not Party to Regional Fisheries Management Organizations

For decades, states have cooperated through regional fisheries management organizations (RFMOs) on the conservation and management of living marine resources on the high seas. Nonmembers, or third states not bound by their decisions, have been an Achilles’ heel. In this article, the legal status of RFMO nonmembers and the freedom of fishing are examined. It is concluded that RFMO nonmembers have concrete obligations with respect to established fisheries subject to RFMO regulation.     

Adapting to the destitute situations: poverty cues lead to short-term choice

Background

Why do some people live for the present, whereas others save for the future? The evolutionary framework of life history theory predicts that preference for delay of gratification should be influenced by social economic status (SES). However, here we propose that the decision to choose alternatives in immediate and delayed gratification in poverty environments may have a psychological dimension. Specifically, the perception of environmental poverty cues may induce people alike to favor choices with short-term, likely smaller benefit than choices with long-term, greater benefit.

Methodology/Principal Findings

The present study was conducted to explore how poverty and affluence cues affected individuals'' intertemporal choices. In our first two experiments, individuals exposed explicitly (Experiment 1) and implicitly (Experiment 2) to poverty pictures (the poverty cue) were induced to prefer immediate gratification compared with those exposed to affluence pictures (the affluence cue). Furthermore, by the manipulation of temporary perceptions of poverty and affluence status using a lucky draw game; individuals in the poverty state were more impulsive in a manner, which made them pursue immediate gratification in intertemporal choices (Experiment 3). Thus, poverty cues can lead to short-term choices.

Conclusions/Significance

Decision makers chose more frequently the sooner-smaller reward over the later-larger reward as they were exposed to the poverty cue. This indicates that it is that just the feeling of poverty influences intertemporal choice – the actual reality of poverty (restricted resources, etc.) is not necessary to get the effect. Furthermore, our findings emphasize that it is a change of the poverty-affluence status, not a trait change, can influence individual preference in intertemporal choice.     

Adapting to the human world: Dogs’ responsiveness to our social cues

Dogs are more skilful than a host of other species at tasks which require they respond to human communicative gestures in order to locate hidden food. Four basic interpretations for this proficiency surface from distilling the research findings. One possibility is that dogs simply have more opportunity than other species to learn to be responsive to human social cues. A different analysis suggests that the domestication process provided an opening for dogs to apply general cognitive problem-solving skills to a novel social niche. Some researchers go beyond this account and propose that dogs’ co-evolution with humans equipped them with a theory of mind for social exchanges. Finally, a more prudent approach suggests that sensitivity to the behaviours of both humans and conspecifics would be particularly advantageous for a social scavenger like the dog. A predisposition to attend to human actions allows for rapid early learning of the association between gestures and the availability of food.     

Down to the origin: Cdc6 protein and the competence to replicate

In eukaryotes, DNA replication requires the regulated assembly of pre-replicative complexes (pre-RCs) onto DNA during G1 phase. Pre-RCs render the chromatin competent to replicate, yet it is only at the G1-S phase transition that protein-kinase complexes trigger the transition to DNA replication. Central to the formation of pre-RCs and regulation of DNA replication is the Cdc6 protein. Two recent studies have shown that Cdc6 is the long-sought factor that confers the competence to replicate in unfertilized Xenopus eggs.     

Adapting and improving crops: the endless task

The Malthusian prognosis has been undermined by an exponential increase in world food supply since 1960, even in the absence of any extension of the arable area. The requisite increases in yield of the cereal staples have come partly from agronomic intensification, especially of nitrogenous fertilizer use made possible by the dwarfing of wheat and rice, in turn made feasible by herbicide development. Cereal dwarfing also contributed to a marked rise in harvest index and yield potential.<br>Although there is still scope for some further improvement in harvest index and environmental adaptation, it is not apparent how a doubling of yield potential can be achieved unless crop photosynthesis can be substantially enhanced by genetic engineering. Empirical selection for yield has not enhanced photosynthetic capacity to date, but nitrogenous and other fertilizers have done so, and there is still scope for agronomic increases in yield and for new synergisms between agronomy and plant breeding. <br>     

Adapting to the unpredictable: reproductive biology of vertebrates in the Australian wet-dry tropics

In the wet-dry tropics of northern Australia, temperatures are high and stable year-round but monsoonal rainfall is highly seasonal and variable both annually and spatially. Many features of reproduction in vertebrates of this region may be adaptations to dealing with this unpredictable variation in precipitation, notably by (i) using direct proximate (rainfall-affected) cues to synchronize the timing and extent of breeding with rainfall events, (ii) placing the eggs or offspring in conditions where they will be buffered from rainfall extremes, and (iii) evolving developmental plasticity, such that the timing and trajectory of embryonic differentiation flexibly respond to local conditions. For example, organisms as diverse as snakes (Liasis fuscus, Acrochordus arafurae), crocodiles (Crocodylus porosus), birds (Anseranas semipalmata) and wallabies (Macropus agilis) show extreme annual variation in reproductive rates, linked to stochastic variation in wet season rainfall. The seasonal timing of initiation and cessation of breeding in snakes (Tropidonophis mairii) and rats (Rattus colletti) also varies among years, depending upon precipitation. An alternative adaptive route is to buffer the effects of rainfall variability on offspring by parental care (including viviparity) or by judicious selection of nest sites in oviparous taxa without parental care. A third type of adaptive response involves flexible embryonic responses (including embryonic diapause, facultative hatching and temperature-dependent sex determination) to incubation conditions, as seen in squamates, crocodilians and turtles. Such flexibility fine-tunes developmental rates and trajectories to conditions--especially, rainfall patterns--that are not predictable at the time of oviposition.     

Rights Based Fishing: Use Rights versus Property Rights to Fish

The basic economic problem of commercial fisheries can be seen as the absence of property rights to the fish stocks. There are, however, both practical and principal obstacles to applying that solution. Instead, the preferable arrangement appears to be stock control by public agencies, combined with exclusive use rights granted, leased or sold to the fishing industry in order to achieve economic efficiency. Incentives to establish such rights can be found both in government circles and in the industry. Both have a vital role to play; plans to establish use rights such as ITQs have run aground on more than one occasion because of opposition from the industry. Use rights can be seen as a tool for government agencies to achieve economic efficiency in the industry. If desired, these gains can be distributed over a wider public through user fees of some kind. There are few examples, however, of substantial user fees, and the industry has been successful in getting such schemes abolished when they have been put into effect. The experience of Russia and, in particular, Estonia is discussed. The absence of user fees can be explained by the need to obtain support for use right schemes from the industry, and the windfall gains from gratis use rights are vehicles for this. Norway and Iceland are two countries which have put in place use rights, but they have done so in different ways. Iceland has an ITQ system while Norway has fishing concessions combined with individual vessel quotas. Some success appears to have been achieved in both countries with these systems.     

Adapting the stretched sample method from tissue profiling to imaging

The characterization and localization of peptides and proteins in tissues provides information that aids in understanding their function and in characterizing disease states. Over the past decades, the use of MS for the profiling and imaging of biological compounds from tissues has evolved into a powerful modality to accomplish these studies. One recently described sampling approach, the stretched sample method (Monroe, E. B. et al.., Anal. Chem. 2006, 78, 6826-6832), places a tissue section onto an array of glass beads embedded on a Parafilm M membrane. When the membrane is stretched, it separates the tissue section into thousands of cell-sized pieces for tissue profiling by MALDI-MS. The physical separation between beads eliminates analyte redistribution during matrix application and allows long analyte extraction periods without loss of spatial resolution. Here, we enhance this sampling approach by introducing algorithms that enable the reconstruction of ion images from these stretched samples. As the first step, a sample-tailored data acquisition method is devised to obtain mass spectra exclusively from the beads, thereby reducing the time, instrument resources, and data handling required for such MS imaging (MSI) experiments. Next, an image reconstruction algorithm matches data acquired from the stretched sample to the initial bead locations. The efficacy of this method is demonstrated using peptide-coated beads with known peptide distributions and appears well-suited to the MSI of heterogeneous tissue samples.     

Adapting the LCA framework to environmental assessment in land planning

Purpose

Since the implementation of the European directive (EC/2001/42) on strategic environmental assessment, an ex ante evaluation has become mandatory for plans and programs. This requirement could have significant consequences for the environment. Local authorities, who are in charge of land planning issues, must therefore conduct such assessments. However, they are faced with lack of uniform methodology. The aim of this paper is thus to propose a methodological framework for the required environmental assessment stages in land planning.

Methods

Life cycle assessment (LCA) has been identified as a promising tool to perform environmental assessment at a meso-level (i.e., territories). Yet, the standardized LCA framework has never been used for assessing the environmental impacts of a territory as such, which can be explained by the complexity that its application would involve. Four major methodological bottlenecks have been identified in this paper, i.e., (1) functional unit definition, (2) boundary selection, (3) data collecting, and (4) the refinement of the life cycle impact assessment phase in order to provide useful indicators for land planning. For each of these challenges, recommendations have been made to adapt the analytical framework of LCA.

Results and discussion

A revised framework is proposed to perform LCA of a territory. One of the major adaptations needed concerns the goal and scope definition phase. Henceforth, the association of a territory and the studied land planning scenario, defined by its geographical boundaries and its interactions with other territories, will be designated as the reference flow in LCA. Consequently, two kinds of indicators will be determined using this approach, i.e., (1) a vector of environmental impacts generated (conventional LCA) and (2) a vector of land use functions provided by the territory for different stakeholders (e.g., provision of work, recreation, culture, etc.). This revised framework has been applied to a theoretical case study in order to highlight its utility in land planning.

Conclusions

This work is a first step in the adaptation of the LCA framework to environmental assessment in land planning. We believe that this revised framework has the potential to provide relevant information in decision-making processes. Nonetheless, further work is still needed to broaden and deepen this approach (i.e., normalization of impacts and functions, coupled application with GIS, uncertainties, etc.).