Dung removal increases under higher dung beetle functional diversity regardless of grazing intensification.

Dung removal by macrofauna such as dung beetles is an important process for nutrient cycling in pasturelands. Intensification of farming practices generally reduces species and functional diversity of terrestrial invertebrates, which may negatively affect ecosystem services. Here, we investigate the effects of cattle-grazing intensification on dung removal by dung beetles in field experiments replicated in 38 pastures around the world. Within each study site, we measured dung removal in pastures managed with low- and high-intensity regimes to assess between-regime differences in dung beetle diversity and dung removal, whilst also considering climate and regional variations. The impacts of intensification were heterogeneous, either diminishing or increasing dung beetle species richness, functional diversity, and dung removal rates. The effects of beetle diversity on dung removal were more variable across sites than within sites. Dung removal increased with species richness across sites, while functional diversity consistently enhanced dung removal within sites, independently of cattle grazing intensity or climate. Our findings indicate that, despite intensified cattle stocking rates, ecosystem services related to decomposition and nutrient cycling can be maintained when a functionally diverse dung beetle community inhabits the human-modified landscape.

Population structure of Phanaeus vindex (Coleoptera: Scarabaeidae) in SE Michigan.

Until now, little is known about the population structure and mobility of temperate dung beetles including the rainbow scarab, Phanaeus vindex (MacLeay 1819), although this knowledge is essential for their conservation as pastures become increasingly rare and the landscape fragmented by monocultures and urbanization. Here, we estimated population size, longevity, and dispersal within and between pastures. For 3 yr, we life-trapped beetles every week on 2 adjacent farms in SE Michigan, determined their sex, male morph, and size, and marked their elytra with individual tattoo patterns before releasing them. We marked a total of 470 rainbow scarabs of which 14 were recaptured once and 2 were recaptured twice. The sex ratio was not significantly sex-biased but fluctuated between months with no apparent uniformity between years. While the minor to major male ratios were unbiased in 2019 and 2020, they were marginally minor-biased in 2021. The gross population estimates for the 2 farms were 458-491 and 217 rainbow scarabs, respectively. Beetles traveled distances of up to 178 m within farms. No beetles dispersed between farms. One large female was recaptured after 338 days documenting the first cold hardiness and long lifespan of a cold-temperate dung beetle species in the wild. The low population estimates on both farms indicate 2 vulnerable populations with no or extremely limited connectivity. Supplementary funding for the land stewardship of small-scale cattle farmers could stabilize populations of native dung beetles and maintain their ecosystem services.

Attractiveness of Cattle Dung to Coprophilous Beetles (Coleoptera: Scarabaeoidea and Sphaeridiinae) and Their Segregation During the Initial Stages of the Heterotrophic Succession on a Pasture in Southeast Michigan.

Only a few mostly older studies analyzed the heterotrophic succession of dung beetles in the Midwestern United States. Such studies are needed to track the impacts of the climate crisis on heterotrophic succession and the associated decomposition processes that are central to soil fertility and carbon sequestration. The current study closes this knowledge gap and provides an easy and efficient method to estimate the relative attractiveness of individual dung pads during heterotrophic succession. The dung beetle community of Carpenter Farm in Adrian, Southeast Michigan was sampled for an entire year, including the winter months, using 15 pitfall traps baited with fresh cow manure. Samples were collected after 48 h and again after 72 h exposure time from the bucket content while leaving the bait unhampered. Eighty-four percent of all beetles were caught in the early sample, but only 6 species were missing in the later sample. A cluster analysis based on Pianka's niche overlap identified a statistically higher mean overlap than expected by chance in a null model (model RA3) and divided the species community clearly into three clusters separating most relocators from most dwellers. Despite using a different method, my results confirmed the successional position of most previously described species and added data for several species with poor or unknown successional state. The successional segregation between dwellers and relocators discovered by the cluster analysis was paralleled by a significantly larger body size of relocators across taxonomic groups as compared to dwellers.

Interactions between amyloid precursor protein-like (APPL) and MAGUK scaffolding proteins contribute to appetitive long-term memory in Drosophila melanogaster.

Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, we previously showed that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). In the present study, we show that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the α'/ß' Kenyon cells. Using a biochemical approach, we identify the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, we show that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the α'/ß' neurons. Finally, using a double RNAi approach, we demonstrate that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, our results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species.

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.

In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon.

Variability of behavioral chronotypes of 16 mammalian species under controlled conditions.

Human chronotypes (differences in preference for early or late rising each day) have been extensively studied in recent years, but no attempt has been made to compare human chronotypes with the chronotypes of other animal species. We evaluated behavioral chronotypes in 16 mammalian species along a body size gradient of five orders of magnitude (from mice to cattle). Individuals of all species were studied under a 12L:12D photoperiod in a thermoneutral environment with food and water available at all times. Rhythms of locomotor activity were analyzed for onset time, acrophase, and robustness. Neither of these rhythmic parameters was significantly related to body size, but onset time and acrophase varied considerably from species to species, thus characterizing diurnal and nocturnal species. Chronotype spreads ranged from less than an hour in sheep to almost 24h in cats, thus extending both below and above the human chronotype spread of 6h. The variability of chronotype (as quantified by the standard deviation of group means) was much larger between species than within species and also larger between individuals of a species than within individuals on consecutive days. These results help situate the matter of human chronotypes within the broader context of variability in the phase angle of entrainment of circadian rhythms in animals.

Daily Activity and Nest Occupation Patterns of Fox Squirrels (Sciurus niger) throughout the Year.

The authors investigated the general activity and nest occupation patterns of fox squirrels in a natural setting using temperature-sensitive data loggers that measure activity as changes in the microenvironment of the animal. Data were obtained from 25 distinct preparations, upon 14 unique squirrels, totaling 1385 recording days. The animals were clearly diurnal, with a predominantly unimodal activity pattern, although individual squirrels occasionally exhibited bimodal patterns, particularly in the spring and summer. Even during the short days of winter (9 hours of light), the squirrels typically left the nest after dawn and returned before dusk, spending only about 7 hours out of the nest each day. Although the duration of the daily active phase did not change with the seasons, the squirrels exited the nest earlier in the day when the days became longer in the summer and exited the nest later in the day when the days became shorter in the winter, thus tracking dawn along the seasons. During the few hours spent outside the nest each day, fox squirrels seemed to spend most of the time sitting or lying. These findings suggest that fox squirrels may have adopted a slow life history strategy that involves long periods of rest on trees and short periods of ground activity each day.

A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity.

The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]. PIKfyve function is required for homoeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the amyloid precursor protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. In the present study, we utilize this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP intracellular domain (AICD) to the HIV TAT domain, a cell-permeable peptide allowing proteins to penetrate cells. The resultant TAT-AICD fusion protein is cell permeable and triggers an increase in PI(3,5)P2 Using the PI(3,5)P2 specific GFP-ML1Nx2 probe, we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT-AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the AICD activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease.

The amyloid precursor protein (APP) binds the PIKfyve complex and modulates its function.

Phosphoinositides are important components of eukaryotic membranes that are required for multiple forms of membrane dynamics. Phosphoinositides are involved in defining membrane identity, mediate cell signalling and control membrane trafficking events. Due to their pivotal role in membrane dynamics, phosphoinositide de-regulation contributes to various human diseases. In this review, we will focus on the newly emerging regulation of the PIKfyve complex, a phosphoinositide kinase that converts the endosomal phosphatidylinositol-3-phosphate [PI(3)P] to phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2)], a low abundance phosphoinositide of outstanding importance for neuronal integrity and function. Loss of PIKfyve function is well known to result in neurodegeneration in both mouse models and human patients. Our recent work has surprisingly identified the amyloid precursor protein (APP), the central molecule in Alzheimer's disease aetiology, as a novel interaction partner of a subunit of the PIKfyve complex, Vac14. Furthermore, it has been shown that APP modulates PIKfyve function and PI(3,5)P2 dynamics, suggesting that the APP gene family functions as regulator of PI(3,5)P2 metabolism. The recent advances discussed in this review suggest a novel, unexpected, ß-amyloid-independent mechanism for neurodegeneration in Alzheimer's disease.

APP controls the formation of PI(3,5)P(2) vesicles through its binding of the PIKfyve complex.

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer's disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein-protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer's disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer's disease.

AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust.

Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbs-positive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

The Amyloid Precursor Protein Controls PIKfyve Function.

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

Septin6 and Septin7 GTP binding proteins regulate AP-3- and ESCRT-dependent multivesicular body biogenesis.

Septins (SEPTs) form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB) biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport)-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies.

Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs.

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.

A novel role for retromer in the control of epithelial cell polarity.

The establishment and maintenance of epithelial cell polarity is essential throughout the development and adult life of all multicellular organisms. A key player in maintaining epithelial polarity is Crumbs (Crb), an evolutionarily conserved type-I transmembrane protein initially identified in Drosophila. Correct Crb levels and apical localization are imperative for its function. However, as is the case for many polarized proteins, the mechanisms of its trafficking and strict apical localization are poorly understood. To address these questions, we developed a liposome-based assay to identify trafficking coats and interaction partners of Crb in a native-like environment. Thereby, we demonstrated that Crb is a cargo for Retromer, a trafficking complex required for transport from endosomes to the trans-Golgi-network. The functional importance of this interaction was revealed by studies in Drosophila epithelia, which established Retromer as a novel regulator of epithelial cell polarity and verified the vast potential of this technique.

Bidirectional transport between the trans-Golgi network and the endosomal system.

The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.

The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.

Early endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150(glued) component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

The V-ATPase in Paramecium: functional specialization by multiple gene isoforms.

The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport.

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment.

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.