CAP1 binds and activates adenylyl cyclase in mammalian cells

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

CAP/srv2 was originally identified in yeast biochemically as an adenylyl cyclase–associated protein (1) and genetically as a suppressor of the hyperactive Ras2-V19 allele (2). CAP/srv2-deficient yeast cells are unresponsive to active Ras2, and adenylyl cyclase activity is no longer regulated by Ras2 in these cells (1, 2), indicating the involvement of CAP/srv2 in the Ras/cyclase pathway. However, some mutant CAP/srv2 alleles presented phenotypes not observed in strains with impaired Ras/cyclase pathway (1–3), indicating the existence of Ras/cyclase-independent functions downstream of CAP/srv2. These two phenotype groups, that is, Ras/cyclase-linked and Ras/cyclase-independent, could be suppressed by expression of an N-terminal half and a C-terminal half of CAP/srv2, respectively (4). Subsequent studies showed that the C-terminal half of CAP/srv2 was able to bind monomeric G-actin (5–8) and other actin regulators establishing a role in F-actin dynamics (9–16). Thus, CAP/srv2 is a bifunctional protein with an N-terminal domain involved in Ras/cyclase regulation and a C-terminal domain involved with F-actin dynamics regulation (16–18).CAP1 is structurally conserved in all eukaryotes (18–22); however, their functions are not. Expression of the closely related Schizosaccharomyces pombe cap or mammalian CAP1 in yeast can only suppress the phenotypes associated with deletion of CAP/srv2’s C-terminal but not its N-terminal domain (19, 20, 22), suggesting that only the F-actin dynamics function was conserved while the Ras/cyclase regulation diverged early on in evolution (16–18). CAP/srv2’s N-terminal 1 to 36 domain was sufficient for cyclase binding in yeast involving a conserved RLE motif with predicted coiled-coil folding (23). Interestingly, this domain is also involved in CAP1 oligomerization both in yeast and mammalian cells (24–26), where it purifies as a high-molecular complex of ∼600 kDa consistent with a 1:1 stoichiometric CAP1-actin hexameric organization (12, 25, 27, 28). Importantly, removal of this domain disrupted CAP1 oligomerization, reduced F-actin turnover in vitro and caused defects in cell growth, cell morphology, and F-actin organization in vivo (24, 29). However, whether the conserved RLE motif in mammalian CAP1 interacts with other coiled-coil–containing proteins is for the moment unknown.Ras2-mediated cyclase regulation in yeast requires its farnesylation (30–32). However, the lipid target involved was not identified in the original studies. We have recently shown that mammalian CAP1 interacts with the small GTPase Rap1. The interaction involves Rap1’s C-terminal hypervariable region (HVR) and its lipid moiety in a geranylgeranyl-specific manner; that is, neither the closely related Ras1 nor engineered farnesylated Rap1 interacted with CAP1 (33). Thus, we raised the question whether CAP1-Rap1, similar to CAP/srv2-Ras2 in yeast, plays a role in cAMP dynamics in mammalian cells.In this study, we report that CAP1 binds to and activates mammalian adenylyl cyclase in vitro. The interaction involves CAP1’s conserved RLE motifs and cyclase’s conserved catalytic subdomains (e.g., C1a and/or C2a). Most importantly, we show that both CAP1 and Rap1 modulate cAMP dynamics in live cells and are critical players in cAMP-dependent proliferation.     

Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)–based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

Trace metals like iron (Fe), copper (Cu), manganese (Mn), and zinc (Zn) play a crucial role as cofactors, providing a range of chemical capabilities to enzymes central to life. Metals also provide structural stability to proteins (1, 2) and enable the catalysis of essential metabolic reactions by providing functional groups that are not readily available via the side chains of amino acids. Consequently, trace metals are required in ∼40% of all enzymes as part of their catalytic centers (3, 4). Individual enzymes are often optimized to utilize a specific metal cofactor, with respect to the chemical utility of that particular trace metal for the catalyzed reaction, the metal’s specific requirements for the binding site in the protein (dimensions, charge, coordination preferences), and the availability of the trace metal within the organism’s reach. There is, however, the possibility of mismetalation, largely attributed to protein structural flexibility and somewhat similar ionic radii and coordination preferences of first-row trace metals (5, 6). Enzyme mismetalation can harm the cell directly by loss of function (7, 8), by accumulation of unintended products, or by the production of toxic side products, for example, reactive oxygen species (9). Cells have therefore developed elaborate strategies to facilitate correct metalation, including preassembling metal cofactors (which allows for easier distinction and delivery of specific metals), the use of metallochaperones (removing especially thermodynamically favored elements like Cu/Zn from the accessible, intracellular trace metal pool), and the compartmentalization of trace metal metabolism (adjusting metal concentrations locally in order to direct binding to target proteins) (6, 10, 11).Chlamydomonas reinhardtii is a unicellular green alga that has been widely used as a eukaryotic, photosynthetic reference system, and therefore exploited in our laboratory to study trace metal metabolism. It has a short generation time (∼6 h), is a facultative heterotroph, and can be grown to high densities (12). Chlamydomonas requires a broad spectrum of metal cofactors to sustain its photosynthetic, respiratory, and metabolic capabilities, with Fe, Cu, Mn, and Zn as the major first-row trace metals involved in these processes. In the last 20 y, studies have revealed a repertoire of assimilatory and distributive transporters in Chlamydomonas, using biochemical and genomics approaches (13–17), discovered mechanisms for metal sparing and recycling to ensure economy (18–21), and identified metal storage sites (2, 22–24).Storage sites are crucial components of trace metal homeostasis. The capacity to sequester individual trace metals is important for controlling protein metalation, detoxification in situations of overload, and buffering during metabolic remodeling. Metal storage provides a selective advantage in competitive environments when transition metals are scarce. One well-known storage site is ferritin, a soluble, mostly cytosolic (animals) or mostly plastidic (plants), 24-subunit oligomer that can oxidize ferrous to ferric Fe and store up to ∼4,500 ferric ions in mineralized form in its core (25, 26). The importance of ferritin as an Fe store is well documented in eukaryotes. In Chlamydomonas, the pattern of expression of ferritin is more consistent with a role in buffering Fe during metabolic transitions, for example from phototrophy to heterotrophy during Fe starvation (23, 27). Vacuoles, lysosome-related, and other acidic organelles, are equally important storage organelles in eukaryotes (28–33). In yeast and plants, these organelles can sequester metals for future use (34). Chlorophyte algae employ a set of smaller cytosolic vacuoles, including contractile vacuoles and acidocalcisomes. Contractile vacuoles (CVs) manage the water content in the cytoplasm; in a fresh water alga that is predominantly facing hypotonic environments (35, 36), water is removed from the cell, potentially using potassium (K) and/or chloride (Cl) (37) to generate an osmotic gradient to attract water to the CV. Acidocalcisomes are lysosome-related organelles in the cytosol, defined by their low pH and high levels of calcium and polyphosphate (polyP) content (38, 39). In Chlamydomonas and other green algae this organelle may also contain K (40, 41). Acidocalcisomes can be identified as electron-dense granules by transmission electron microscopy (TEM) or by utilizing specific probes targeting either the low pH environment or the specific elemental makeup of the compartment (24, 42–44). In addition, acidocalcisomes have been visualized using their unique elemental signature by mass spectrometry (MS)-based imaging techniques, specifically nanoscale secondary ion mass spectrometry (nanoSIMS), or X-ray fluorescence microscopy (XFM) (45, 46). These methods demonstrated that, at least in Chlamydomonas, the acidocalcisome can house high amounts of Cu and Mn in excess conditions (24, 47), making it a prime candidate for Fe storage as well.XFM is a synchrotron-based technique that utilizes X-rays to produce photons in the object to be visualized. The energies of these fluorescent photons are element specific and can be used to determine elemental distribution and concentrations in whole cells or subcellular compartments (48–50). High energy X-rays (>10 keV) penetrate biological material deep enough so that no sectioning is required. True elemental distributions are obtained when metabolic states are preserved rapidly using either vitrification or chemical fixation. XFM has previously been utilized to determine the elemental content of vacuoles of phagocytes infected with various Mycobacterium species (51), to demonstrate transient zinc relocation to the nucleus during macrophage differentiation (52) and the mobilization of Cu during angiogenesis (53).In this work, we employed XFM with sub-100-nm spatial resolution to identify an Fe storage site in Chlamydomonas, determine the spatial distribution of multiple essential trace elements in chlorophyte algae, and quantify acidocalcisomal metal content in single cells in situations of Fe and Cu overaccumulation. We took advantage of a Chlamydomonas vacuolar transporter chaperone (vtc1) mutant (defective in polyP synthesis and hence also Ca content) (43, 47) to distinguish the role of polyP and Ca in acidocalcisome Fe sequestration. XFM also enabled the comparison of single-cell trace metal quotas with bulk quantification of Cu and Fe in corresponding cell cultures, allowing us to distinguish the nutritional state for Cu and Fe in individual cells.     

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5′ splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 ({"type":"entrez-nucleotide","attrs":{"text":"NM_018077.2","term_id":"187960108","term_text":"NM_018077.2"}}NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Ribosome biogenesis (RB) is the essential cellular process in which the complex macromolecular ribosomal machinery is manufactured and assembled, enabling protein translation (1–4). Both ribosomal RNA (rRNA) and ribosomal protein (RP) components must be correctly synthesized, processed, modified, folded, translocated, and ultimately joined in the cytoplasm to engage in global protein synthesis (1–3). For eukaryotes, four rRNA molecules (1, 5) and about 80 RPs (1, 6, 7) form the core of the mature small (40S) and large (60S) ribosomal subunits. The demand for ribosomes during the cell cycle is immense: in a growing yeast cell, more than 30 ribosomes are synthesized per second (8), while in a growing HeLa cell, this figure balloons to 125 ribosomes per second (9). Over 200 trans-acting assembly factors are necessary to achieve the fast and accurate ribosome assembly required to meet this tremendous cellular translational demand (1).Given that up to 80% of cellular metabolism is devoted to RB (10), it is unsurprising that defects in RB factors are causative of a class of human diseases known as ribosomopathies (1, 11–16). Though not fully understood, tissue-specific defects are the hallmark of ribosomopathies (11, 17). Tissues formed from hematopoietic or neural crest cell lineages are disproportionately affected, resulting in anemia, neutropenia, and leukemia, bone marrow failure diseases including Diamond–Blackfan Anemia (DBA) (18–23) and Shwachman–Diamond syndrome (24–26), craniofacial, dermatological, and neurological diseases including Treacher Collins syndrome (27–29) and postaxial acrofacial dysostosis (30), and alopecia, neurologic defects, and endocrinopathy (ANE) syndrome (31–34).ANE syndrome (OMIM: 612079) (31, 35) is a rare ribosomopathy defined by heterogeneous clinical features of variable severity including alopecia, neurological deformities, intellectual disability, and hormonal deficiencies with pubertal delay. In the only ANE syndrome case report published to date, Nousbeck and coworkers studied five brothers of consanguineous parentage with variable ANE syndrome features, finding that ANE syndrome patient tissue samples had quantifiably fewer ribosomes and qualitatively dysmorphological rough endoplasmic reticula versus healthy control samples (31). All five patients were found to carry a homozygous missense variant (p.(Leu351Pro); L > P) in RBM28 (31), a known essential 60S assembly factor orthologous to yeast Nop4 (36–39). Follow-up studies further defined the clinical extent of endocrinopathy (32) and the biochemical mechanisms of hair and skin defects (33) and of inhibited ribosome biogenesis (34, 40) due to impaired function of RBM28 or its yeast homolog, Nop4. However, due to the rarity of the disease and lack of sufficient animal model studies, further investigation of ANE syndrome has been limited.We report a female pediatric patient in the second family of ANE syndrome to date, unrelated to the family in the original case report (31). The ANE syndrome patient has a clinical presentation consistent with the definition of ANE syndrome but possesses differing genetic variants and molecular pathology. Using in vivo techniques, we demonstrate that the patient’s compound heterozygous splicing variants in RBM28 create one hypomorphic (ΔE5) and one null (ΔE8) allele with respect to overall growth and 60S pre-rRNA processing functions. By elucidating the pathology of an ANE syndrome patient, our results bolster and extend our understanding of this rare ribosomopathy and reinforce the importance of proper nucleolar function in human health and disease.     

Towards the molecular architecture of the peroxisomal receptor docking complex

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

Peroxisomes are organelles present nearly ubiquitously in eukaryotic cells, ranging from unicellular yeasts to multicellular organisms, such as plants and humans. Beside β-oxidation of fatty acids as a main conserved function of peroxisomes, a broad range of additional metabolic functions is linked to this organelle, underscored by severe and frequently lethal phenotypes of human disorders (1, 2). These organelles do not contain DNA and thus all peroxisomal matrix proteins are encoded in the nucleus and synthesized on free polyribosomes in the cytosol. Subsequently, matrix proteins are targeted to the organelle by peroxisomal import receptors (3). A remarkable feature of peroxisomes is that unlike the transport of unfolded polypeptides across the membranes of the endoplasmic reticulum and mitochondria, they can import already folded, cofactor-bound, and even oligomeric proteins (4, 5). This transport is highly selective and mediated by specific import sequences known as peroxisomal targeting signals (PTSs) (6, 7). Peroxisomal matrix proteins equipped with either a carboxyl-terminal PTS1 or an amino-terminal PTS2, are recognized and bound in the cytosol by the import receptor Pex5p or Pex7p, respectively (8, 9). A peroxisomal membrane-associated complex consisting of Pex13p, Pex14p, and Pex17p in yeast allows docking of the cargo-loaded receptor (10–14). This primary interaction of the cargo-loaded receptor with the docking complex induces the formation of a transient and highly dynamic import pore, necessary for the translocation of the cargo across the peroxisomal membrane (15–17). How translocation and release of the cargo are realized in detail still remains enigmatic but it has been previously shown that the receptor is exported from the peroxisomal membrane in an ubiquitin- and ATP-dependent manner, a process that is discussed to provide the driving force for cargo import according to the export-driven import model (18–20).The receptor–docking complex is of major importance for peroxisomal matrix protein import, as it provides a binding platform for newly formed receptor–cargo complexes at the peroxisomal membrane. Both Pex13p and Pex14p are peroxisomal membrane proteins providing several binding sites for the import receptors Pex5p and Pex7p (16). Docking of the Pex5p–PTS1 protein complex at the peroxisome membrane is supposed to occur at Pex14p (21, 22). Pex17p is tightly associated with Pex14p (23), but its precise function remains unknown. Although Pex17p is part of the docking complex in yeast, it does not significantly contribute to the assembly of the Pex13p/Pex14p subcomplex (15, 23, 24), and its counterpart in higher eukaryotes has not yet been identified. However, Pex17p is essential for peroxisomal import of both PTS1 and PTS2 proteins (14). Strikingly, both import receptors, Pex5p and Pex7p, associate with the docking complex (Pex13p/Pex14p) in absence of Pex17p, but with decreased efficiency (24).Furthermore, albeit a close association between the core components of the docking complex (Pex13p/Pex14p) is important for matrix protein import (25), there are several lines of evidence that Pex13p is not a permanent component of the peroxisomal docking complex or the import pore (10, 26) and interestingly, an assembly between the receptor Pex5p and the docking component Pex14p in absence of Pex13p is capable per se of forming a large transient channel at the peroxisome membrane (15).However, little is known about the molecular mechanism underlying the primary docking and subsequent translocation events, largely because structures of the higher-order assemblies are not available. Here, using cryo-electron microscopy single particle analysis (cryoEM SPA) and cryo-electron tomography (cryoET) combined with cross-linking and native mass spectrometry (MS), we set out to characterize the overall architecture of the yeast Pex14p/Pex17p complex.     

Conditions and extent of volatile loss from the Moon during formation of the Procellarum basin

Rocks from the lunar interior are depleted in moderately volatile elements (MVEs) compared to terrestrial rocks. Most MVEs are also enriched in their heavier isotopes compared to those in terrestrial rocks. Such elemental depletion and heavy isotope enrichments have been attributed to liquid–vapor exchange and vapor loss from the protolunar disk, incomplete accretion of MVEs during condensation of the Moon, and degassing of MVEs during lunar magma ocean crystallization. New Monte Carlo simulation results suggest that the lunar MVE depletion is consistent with evaporative loss at 1,670 ± 129 K and an oxygen fugacity +2.3 ± 2.1 log units above the fayalite-magnetite-quartz buffer. Here, we propose that these chemical and isotopic features could have resulted from the formation of the putative Procellarum basin early in the Moon’s history, during which nearside magma ocean melts would have been exposed at the surface, allowing equilibration with any primitive atmosphere together with MVE loss and isotopic fractionation.

Returned samples of basaltic rocks from the Moon provided evidence decades ago that the Moon is depleted in volatile elements compared to the Earth (1), with lunar basalt abundances of moderately volatile elements (MVEs) being ∼1/5 that of terrestrial basalt abundances for alkali elements and ∼1/40 for other MVE, such as Zn, Ag, In, and Cd (2). The theme of lunar volatiles thus seemed settled. Yet, the unambiguous detection in 2008 of lunar indigenous hydrogen and other volatile elements, such as F, Cl, and S in pyroclastic glasses (3), heralded a new era of research into lunar volatiles, overturning the decades-old paradigm of a bone-dry Moon (e.g., refs. 4 and 5). Here, we define volatile elements as those with 50% condensation temperatures below these of the major rock-forming elements Fe, Mg, and Si (6). This paradigm shift was accompanied by new measurements of volatile stable isotope compositions (e.g., H, C, N, Cl, K, Cr, Cu, Zn, Ga, Rb, and Sn) in a wealth of bulk lunar samples (7–18) and in the mineral phases and melt inclusions they host (19–28). These studies have shown that the stable isotope compositions of most MVEs (e.g., K, Zn, Ga, and Rb) are enriched in their heavier isotopes with respect to the bulk silicate Earth (BSE) (9, 11, 13–15, 17). Such heavy isotope enrichment is associated with elemental depletion, which has been variously attributed to liquid–vapor exchange and vapor loss from the protolunar disk (17, 18), incomplete accretion of MVEs during condensation of the Moon (13, 29, 30), and degassing of these elements during lunar magma ocean crystallization (9, 11, 14, 15, 25, 31). Almost all these hypotheses have typically assumed that the MVE depletions and associated MVE isotope fractionations are relevant to the whole Moon. However, our lunar sample collections are biased, as all Apollo and Luna returned samples come from the lunar nearside from within or around the anomalous Procellarum KREEP Terrane (PKT) region (e.g., ref. 32), where KREEP stands for enriched in K, REEs, and P. Barnes et al. (26) proposed that the heavy Cl isotope signature measured in KREEP-rich Apollo samples resulted from metal-chloride degassing from late-stage lunar magma ocean melts in response to a large crust-breaching impact event, spatially associated with the PKT region, which facilitated exposure of these late-stage melts to the lunar surface. Here, we further investigate whether a localized impact event could have been responsible for the general MVE depletion and heavy MVE isotope enrichment measured in lunar samples.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

Stage-specific overcompensation,the hydra effect,and the failure to eradicate an invasive predator

As biological invasions continue to increase globally, eradication programs have been undertaken at significant cost, often without consideration of relevant ecological theory. Theoretical fisheries models have shown that harvest can actually increase the equilibrium size of a population, and uncontrolled studies and anecdotal reports have documented population increases in response to invasive species removal (akin to fisheries harvest). Both findings may be driven by high levels of juvenile survival associated with low adult abundance, often referred to as overcompensation. Here we show that in a coastal marine ecosystem, an eradication program resulted in stage-specific overcompensation and a 30-fold, single-year increase in the population of an introduced predator. Data collected concurrently from four adjacent regional bays without eradication efforts showed no similar population increase, indicating a local and not a regional increase. Specifically, the eradication program had inadvertently reduced the control of recruitment by adults via cannibalism, thereby facilitating the population explosion. Mesocosm experiments confirmed that adult cannibalism of recruits was size-dependent and could control recruitment. Genomic data show substantial isolation of this population and implicate internal population dynamics for the increase, rather than recruitment from other locations. More broadly, this controlled experimental demonstration of stage-specific overcompensation in an aquatic system provides an important cautionary message for eradication efforts of species with limited connectivity and similar life histories.

Theoretical population models can produce counterintuitive predictions regarding the consequences of harvest or removal of predatory species. These models show that for simple predator-prey systems, there can be positive population responses to predator mortality resulting from harvest for fisheries or population management, which can create an increased equilibrium level of that predator species (1–5). Among these mortality processes is the “hydra effect,” named after the mythical multi-headed serpent that grew two new heads for each one that was removed (6, 7). This counterintuitive outcome can be driven by a density-dependent process known as overcompensation. The hydra effect typically refers to higher equilibrium or time-averaged densities in response to increased mortality, typically involving consumer populations undergoing population cycles. Population increases in response to mortality can be the result of stage-specific overcompensation, which involves an increase in a specific life history stage or a size class following increased mortality. The first analysis of overcompensatory responses to mortality did not depend on stage specificity and was applied initially to fisheries harvests (1). Subsequent models have included stage specificity and have been applied to a broad range of systems in which species have been harvested for consumption or removed for population control of non-native species (4, 5, 8–15).Theory suggests that overcompensation in response to harvest or removal can occur for a variety of reasons, including 1) reduced competition for resources and increased adult reproduction rates, 2) faster rates of juvenile maturation or greater success in reaching the adult stage, and 3) increased juvenile or adult survival rates (1–7). An increase in reproductive output in response to reduced adult density can be the result of a reduction in resource competition (SI Appendix, Fig. S1).While there is substantial evidence that conditions that could produce density-dependent overcompensation occur frequently, evidence for overcompensation in natural populations is rare. For only a few populations do we have the long-term demographic data collected over a sufficiently long duration and for population densities over a wide enough range to detect this effect. Unfortunately, recent reviews of population increases in response to increased mortality do not include field studies with explicit controls for removals (13–17).There are examples of density-dependent overcompensation from field populations (4, 13–15), as well as a larger number of studies from the laboratory and greenhouse typically involving plant and insect populations (18–22). Among the field examples is a population control program for smallmouth bass in a lake in upstate New York, which paradoxically resulted in greater bass abundance, primarily of juveniles, after 7 y of removal efforts (23, 24). Another field study in the United Kingdom showed that perch populations responded similarly when an unidentified pathogen decimated adults (25). Other programs that attempted to remove invasive fishes, including pikeperch in England (26), brook trout in Idaho (27), and Tilapia in Australia (28), showed similar results. However, although many of these examples involved well-executed studies with substantial field data, none had explicit controls for removal, such as comparable populations without harvest (or disease). Thus, despite the support of current theory in these studies, the contribution of external factors to observed population responses to harvest remains uncertain. To date, we are unaware of any experimental studies with comparable controls in a field population that demonstrates overcompensation in a single species (13–15).     

Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein–TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

Lipid droplets (LDs) are the intracellular organelles responsible for fat accumulation (1). As such, they play a central role in lipid and cellular metabolism (1–4), and they are crucially involved in metabolic diseases such as lipodystrophy and obesity (5–7).Formation of LDs occurs in the endoplasmic reticulum (ER), where neutral lipids (NLs), namely triglycerides (TG) and cholesteryl esters, constituting the core of LDs are synthesized by acyltransferases that are essential for LD formation (8). The current model of LD formation posits that NLs are stored between the two leaflets of the ER bilayer, where they aggregate in nascent oblate lens-like structures with diameters of 40 to 60 nm (9) before complete maturation and budding toward the cytosol (10–13).Recent experiments suggest that LDs form at specific ER sites marked by the protein seipin (14) upon arrival of its interaction partner protein promethin/LDAF1 (lipid droplet organization [LDO] in yeast) (15–19). These recent observations confirm previous works showing that seipin, in addition to modulating LD budding and growth (14, 19–21) and LD–ER contacts (22, 23), is also a major player in the early stages of LD formation, as deletion of seipin leads to TG accumulation in the ER and a delay in the formation of, possibly aberrant, LDs (20, 24).The role of seipin in LD formation is potentially coupled to its function in regulating lipid metabolism (25, 26) and notably that of phosphatidic acid (PA) (27–31). Recently, seipin-positive ER loci have been shown to be part of a larger protein machinery that also includes membrane and lipid remodeling proteins of the TG synthesis pathway (32), most notably, Lipin (Pah1 in yeast) and FIT proteins (Yft2 and Scs3 in yeast), for which PA is either a known substrate (Lipin/Pah1) (33) or a likely one (FIT/Yft2/Scs3) (34).Despite this thorough characterization of the cellular role of seipin in LD formation, the molecular details of its mechanism remain mostly unclear. Recently, the structure of the luminal part of the seipin oligomer has been solved at 3.7 to 4.0 Å resolution using electron microscopy (27, 35), paving the way for the investigation of the relationship between its three-dimensional structure and its mode of action. These studies revealed that the luminal domain of seipin consists of an eight-stranded beta sandwich, together with a hydrophobic helix (HH), positioned toward the ER bilayer. Notably, the seipin oligomer assembles into a ring-like architecture, an unconventional assembly in lipid bilayers that rather resembles the shape of microbial pore-forming assemblies (36) or GroEL-GroES chaperones (37, 38).From a stochiometric point of view, both fluorescence and electron microscopy data are consistent with the presence of a single seipin oligomer per nascent LD (14, 15). Hence, the structure of the luminal part of seipin is consistent with two proposed modes of action: seipin could mark the sites of LD formation by controlling TG flow in and out of the nascent droplet (14), or, alternatively, seipin could help recognize and stabilize preexisting nascent droplets in the ER membrane (20, 21, 39). In both cases, however, the relationship between the role of seipin in LD formation and its ability to regulate lipid metabolism remains unclear.Here, we use coarse-grain (CG) molecular dynamics (MD) simulations to investigate the mechanism of seipin in molecular detail. We find that seipin is able to cluster TG molecules inside its ring-like structure and that both the transmembrane (TM) helices and the luminal domain contribute to this process. Diacylglycerol (DG), the lipid intermediate between TG and PA in the Kennedy pathway, also accumulates around seipin, further promoting the accumulation of TG at very low TG-to-phospholipids ratios. Our data suggest that by accumulating DG and TG molecules, seipin generates ER sites with a specific lipid composition that in turn could promote the sequential recruitment of additional TG- and DG-sensing proteins involved in LD formation, including promethin/LDOs, FIT/Yft2/Scs3, and perilipins.     

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Diet crucially affects the health of all animals (1). Most animals have a gut microbiota that can also affect host health both positively and negatively (2–6). However, understanding of the mechanisms underlying the effects of the gut microbiota remains a major challenge. This is because animals often consume complex or variable diets, and harbor large, multimember microbial communities that can result in many interactions that hinder identification of the factors responsible for particular host responses (2, 6–11). Metaanalyses and multiomic approaches can provide inferential insights on how diet–microbe or microbe–microbe interactions affect hosts (11–18), but functional support can be difficult to generate if proposed mechanisms cannot be studied experimentally (2, 14). Thus, study systems where hosts can be reared on defined diets with or without a microbiota of known composition can significantly advance mechanistic insights by providing the means to control and manipulate dietary, microbial, and environmental variables that potentially affect a given host response (19–21).Mosquitoes are best known as insects that blood feed on humans and other vertebrates. Only adult-stage female mosquitoes blood feed, which is required for egg formation by most species (22). Blood feeding has also led to several mosquitoes evolving into vectors that can transmit disease-causing microbes between hosts (22). In contrast, the juvenile stages of all mosquitoes are aquatic, with most species feeding on detritivorous diets (22–24). Larvae hatch from eggs with no gut microbiota but quickly acquire relatively low-diversity communities from the environment by feeding (25). Most gut community members are aerobic or facultatively anaerobic bacteria in four phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria), although other microbes, such as fungi and apicomplexans, have also been identified (25–39). Gut community composition also commonly varies within and between species as a function of where larvae develop, diet, and other variables (28–30, 32, 34, 40–42).Aedes aegypti has a worldwide distribution in tropical and subtropical regions, and is the primary vector of the agents that cause yellow fever, dengue fever, and lymphatic filariasis in humans (43). Preferentially living in urban habitats, females lay eggs in water-holding containers with microbial communities, and larvae molt through four instars before pupating and emerging as adults (30, 35, 41, 43). Conventionally reared cultures with a gut microbiota are usually maintained in the laboratory under conditions that mimic natural habitats with rearing temperatures of 25 to 28 °C and a 12- to 16-h light: 8- to 12-h dark photoperiod (44–46). Most insects that require microbial partners for survival live on nutrient-poor diets where microbes provision nutrients that cannot be synthesized or produced in sufficient abundance by the host (3). Mosquito larvae can experience resource limitations in the field (23–25), but in the laboratory are reared on undefined, nutrient-rich diets, such as rodent chow, fish food flakes, or mixtures of materials like liver powder, fish meal, and yeast extract (44–46). Nonetheless, our previous studies indicated that axenic A. aegypti as well as other species consume but fail to grow beyond the first instar when fed several diets that support the development of nonsterile, conventionally reared larvae (30, 47–49). Escherichia coli and several other bacteria identified as gut community members could colonize the gut (producing monoxenic, gnotobiotic larvae) and rescue development, but feeding axenic larvae dead bacteria could not (30, 35, 47). The presence of a gut microbiota in conventional and gnotobiotic but not axenic larvae was also associated with midgut hypoxia and activation of several signaling pathways with growth functions (50, 51). Finally, our own previous results using a strain of E. coli susceptible to ampicillin (50), and more recently a method for clearing an auxotrophic strain of E. coli from gnotobiotic larvae (52), both showed that the proportion of individuals that develop into adults correlates with the duration that larvae have living bacteria in their gut.Altogether, the preceding results suggested that A. aegypti and several other mosquitoes require a gut microbiota for development. In contrast, another recent study showed that axenic A. aegypti larvae develop into adults, albeit more slowly than larvae with a gut microbiota, when fed diets comprised of autoclaved bovine liver powder (LP) and brewer’s yeast (Saccharomyces cerevisiae) extract (YE) or autoclaved LP, YE, and E. coli (EC) embedded in agar (53). This latter finding suggests the undefined dietary components used provide factors larvae require for development into adults, whereas a gut microbiota was also required to provide these factors under the conditions in which our own previous studies were conducted. The goal of this study was to identify what these factors are. Toward this end, we first assessed the growth of axenic A. aegypti when fed diets containing autoclaved LP, YE, and EC under different conditions. We then used this information to develop a defined diet that allowed us to systematically manipulate nutrient, microbial, and environmental variables. We report that the instability of riboflavin is a key factor underlying why A. aegypti larvae require a gut microbiota under most conditions experienced in the laboratory and field.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Determinism and contingencies shaped the evolution of mitochondrial protein import

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei. The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

Intracellular endosymbionts lack protein import systems, whereas such systems are a defining feature of mitochondria and plastids, both of which evolved from bacterial endosymbionts (1–3). Today, more than 95% of all mitochondrial proteins are imported from the cytosol, which makes mitochondrial protein import a key process required for mitochondrial biogenesis (4–6). The question of how mitochondrial protein import evolved is therefore central to understand how the endosymbiotic bacterial ancestor of mitochondria converted into an organelle that is genetically integrated into the host cell (7–9).Proteins are targeted to mitochondria by internal or external import signals, the most frequent one of which is the N-terminal presequence found in 60 to 70% of all imported proteins (10, 11). Interestingly, the various mitochondrial import signals are conserved even between highly diverged eukaryotes (6). The import signals are decoded by receptors, which are integral mitochondrial outer membrane (OM) proteins that are associated with the heterooligomeric protein translocase of the OM (TOM complex) (6, 12). Contrary to the core components of the TOM complex (Tom40, Tom22, and Tom7), which are highly conserved in essentially all eukaryotes, these receptors evolved independently in different eukaryotic lineages, even though they recognize the same conserved import signals (6).The best studied prototypical import receptors are Tom20 and Tom70 of yeast, orthologs of which are found in all members of the eukaryotic supergroup of the opisthokonts (13). Tom20 is an N-terminally anchored OM membrane protein, and its cytosolic domain contains a single tetratricopeptide repeat (TPR). Tom20 preferentially recognizes precursor proteins that have N-terminal presequences. It binds to the hydrophobic surface of the presequence and transfers the precursors to the highly conserved Tom22 that functions as a secondary receptor (14–17). Tom70 is the primary receptor for proteins that have multiple membrane spanning domains, such as mitochondrial carrier proteins, but also binds to hydrophobic precursor proteins that have presequences (18–20). Moreover, it has been shown that binding of Tom70 to the mitochondrial presequence-like stretches that are present in the mature part of many precursor proteins increases the import efficiency (21). Tom70 is N-terminally anchored in the membrane. Its large cytosolic domain consists of 11 TPR motifs. The three TPR motifs proximal to the membrane interact with cytosolic Hsp70 or Hsp90, from which Tom70 can receive precursor proteins (22, 23). The remaining eight TPR motifs directly recognize substrate proteins (24, 25). In yeast, Tom20 and Tom70 have partially redundant functions. Tom70 is not essential for growth and respiration. Loss of Tom20 causes a stronger phenotype; it abolishes respiration but is not lethal. Finally, even the deletion of Tom70 and Tom20 does not kill the cells, provided that the secondary receptor Tom22 is still present (15, 26–29).A single import receptor, termed Tom20, is associated with the TOM complex of plant mitochondria. Yeast and plant Tom20 (30) are superficially similar: both have a single transmembrane domain (TMD) and a soluble domain containing one (in yeast) and two TPR motifs (in plants). Furthermore, both proteins have the same domain organization provided that they are aligned in an antiparallel way. Thus, whereas yeast Tom20 is N-terminally anchored, plant Tom20 is a C-terminally anchored protein. This strongly suggests that yeast and plant Tom20, while both being import receptors, have different evolutionary origins (31, 32). Moreover, plants have another TPR domain-containing OM protein, termed OM64, that is not associated with the TOM complex, but implicated in protein import (31, 33).ATOM46 and ATOM69 are the two receptor subunits of the atypical translocase of the OM (ATOM) of trypanosomatids (34). ATOM69 is superficially similar to yeast Tom70. Both have the same molecular mass and multiple TPR-like motifs. ATOM69, in addition, has an N-terminal CS/Hsp20-like domain, which potentially can bind to cytosolic chaperones. Analogous to plant Tom20, ATOM69 is C-terminally membrane-anchored, whereas yeast Tom70 has an N-terminal TMD. ATOM46 also has an N-terminal membrane anchor and a cytosolic armadillo (ARM) repeat domain, a protein–protein interaction module specific for eukaryotes. The cytosolic domains of ATOM69 and ATOM46 were shown to bind a number of different precursor proteins and are essential for normal growth (34). ATOM69 and ATOM46 have been found in all kinetoplastids as well as in euglenoids (35). Except for the TPR domain in ATOM69, the two import receptors of trypanosomes do not resemble the TOM subunits of other species, indicating that they evolved independently from both the yeast and the plant receptors.Recently, an analysis of the TOM complex in Trichomonas vaginalis hydrogenosomes, which are mitochondria-derived hydrogen-producing organelles that lack their own genome (36), identified Tom36 and Tom46 (37). The two proteins are paralogues and consist of an N-terminal CS/Hsp20-like domain, three TPR-like sequences, and a C-terminal membrane anchor, which is reminiscent of trypanosomal ATOM69, although the mass of both hydrogenosomal proteins is much lower than that of ATOM69. Moreover, HHpred analysis, using Tom36 as a query, retrieved ATOM69 as the first hit (37). The cytosolic domains of Tom36 and Tom46 were able to bind hydrogenosomal precursor proteins, suggesting they may function as protein import receptors. However, despite the similarities between ATOM69 and Trichomonas Tom36/Tom46, phylogenetic analysis suggests that they evolved independently of each other, and therefore reflect yet another example of convergent evolution, although a diversification of a common ancestor cannot be ruled out (37).Here, we have investigated the substrate specificity of the trypanosomal import receptors ATOM46 and ATOM69 using inducible RNA interference (RNAi) cell lines and biochemical methods. We could correlate the observed receptor preference with specific features of the recognized substrate proteins, such as the presence of a predicted presequence, average hydrophobicity, and presence of TMDs. Moreover, we devised a method that allows for identification of which trypanosomal precursor proteins can be recognized by heterologous import receptors. Using this method, the mitochondrial proteomes are quantitatively compared between Trypanosoma brucei cell lines lacking either ATOM46 or ATOM69 and with T. brucei cell lines in which ATOM46 or ATOM69 were replaced by either Tom20 from yeast or Tom36 from Trichomonas.     

Conditional destabilization of the TPLATE complex impairs endocytic internalization

In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta.

Endocytosis is an evolutionarily conserved eukaryotic pathway by which extracellular material and plasma membrane (PM) components are internalized via vesicles (1, 2). Clathrin-mediated endocytosis (CME), relying on the scaffolding protein clathrin, is the most prominent and the most studied endocytic pathway (3–5). As clathrin does not interact directly with the PM, nor does it recognize cargoes, adaptor proteins are required to act as essential links between the clathrin coat and the PM (6). In plant cells, material selected for CME is recognized by two adaptor complexes, the adaptor complex 2 (AP-2) and the TPLATE complex (TPC) (7–9). In contrast to TPC, single subunit mutants of AP-2 are viable (7, 8, 10–13) and AP-2 recruitment and dynamics appear to rely on TPC function (8, 14).TPC represents an ancestral adaptor complex, which is however absent in present-day metazoans and yeasts. It was experimentally identified as an octameric complex in Arabidopsis and as a hexametric complex in Dictyostelium (8, 15). Plants, however, are the only eukaryotic supergroup identified so far where TPC is essential for life (8, 15), as knockout or severe knockdown of single subunits of TPC in Arabidopsis leads to pollen or seedling lethality, respectively (8, 13). Two TPC subunits, AtEH1/Pan1 and AtEH2/Pan1, were not associated with the other TPC core components when the complex was forced into the cytoplasm by truncating the TML subunit and did not copurify with the other TSET components in Dictyostelium. This indicates that they may be auxiliary components to the core TPC (8, 15). These AtEH/Pan1 proteins were recently identified as important players in actin-regulated autophagy in plants. AtEH/Pan1 proteins recruit several components of the endocytic machinery to the autophagosomes, and are degraded together with them under stress conditions (16). However, whether this pathway serves to degrade specific cargoes or to regulate the endocytic machinery itself (17), and whether the whole TPC is required for this degradation pathway, remains unclear.Genetic and chemical tools to manipulate endocytosis have been extensively investigated via interfering with the functions of endocytic players, such as clathrin (18–22), adaptor proteins (7, 10–12, 14, 23–25), and dynamin-related proteins (26–30). The chemical inhibitors originally used to affect CME in plants have recently been described to possess undesirable side effects (31) or to affect proteins that are not only specific for endocytosis: for example, clathrin itself, as it is also involved in TGN trafficking (19, 22). The same is true for several genetic tools currently available to affect CME in plants (18, 21, 22, 30). Manipulation of TPC, functioning exclusively at the PM, represents a very good candidate to affect CME more specifically. So far however, there are no chemical tools to target TPC functions or dominant-negative mutants available. Inducible silencing works, but causes seedling lethality and takes several days to become effective (8). The only tools to manipulate TPC function in viable plants consist of knock-down mutants with very mild reduction of expression and consequently similar mild effects on CME (8, 14, 16, 32).     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

Cognitive aging is associated with redistribution of synaptic weights in the hippocampus

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others’ lifespans outlast their “mindspans.”

Aging is the biggest risk factor for Alzheimer’s disease, but many aged individuals nevertheless retain the ability to perform cognitive tasks with young adult (YA)-like competency, and are thus resilient to age-related cognitive decline and dementias (1, 2). The mechanisms of such resilience are unknown, but are thought to involve neural or cognitive reserve, brain network adaptations, or simply the ability to maintain cognitive brain circuits in a YA-like state (3–5). Much of the cellular and functional insight into the concept or risk of/resilience against age-related cognitive impairments has come from animal models of normal/nonpathological aging (6–10). Many of these studies have shown that circuit function abnormalities are associated with behavioral impairments. The cellular and structural bases for such functional aberrations, however, remain largely unknown.Two of the most well-studied cognitive domains that show susceptibility to chronological aging in both rodents and nonhuman primates are working memory and spatial/temporal memory (6–10). Importantly, these cognitive domains engage anatomically distinct neurocognitive systems, with the former relying on prefrontal/orbitofrontal cortical circuits and the latter relying on hippocampal circuitry. Interestingly, although behavioral deficits in these two domains (in the case of rat models of cognitive aging) begin to emerge, worsen, and become increasingly prevalent between 12 and 18 mo of age in most strains (reviewed in refs. 9 and 11), cognitive aging within hippocampus-dependent forms of learning and memory are relatively independent of those that engage the prefrontal/orbitofrontal cortical neural systems (6–9, 12–15).Neither the mechanisms underlying the conservation of memory function across chronological aging nor those contributing to the age-related emergence and exacerbation of memory impairments are clearly understood for either neurocognitive system. It is clear, however, that neither frank neuronal loss (16, 17) nor overall synapse loss (18) contributes to cognitive aging within the medial temporal lobe/hippocampal memory system. Rather, the intriguing idea that has emerged from work in both the hippocampal and the prefrontal/orbitofrontal cortical memory systems is that there are functional alterations in the synaptic connections in individual microcircuits embedded within these larger neuroanatomical systems (6–10, 19–31).Axospinous synapses (including those in hippocampal and cortical circuits) are characterized on the basis of the three-dimensional morphology of their postsynaptic densities (PSDs) (20, 32–34). The most-abundant axospinous synaptic subtype has a continuous, macular, disk-shaped PSD, as compared to the less-abundant perforated synaptic subtype, which has at least one discontinuity in its PSD (34). In addition to differing substantially with regard to relative frequency, perforated and nonperforated synapses also harbor major differences in size and synaptic AMPA-type and NMDA-type receptor expression levels (AMPAR and NMDAR, respectively) (34–38). There is also evidence that perforated and nonperforated synapses are differentially involved in synaptic plasticity (39–44) and in preservation of—or reductions in—memory function during chronological aging (6, 20, 45). Layered onto these general distinctions between perforated and nonperforated synapses are more specific differences in their characteristics when considered within neural circuits. For example, perforated synapses have a stronger and more consistent influence on neuronal computation within hippocampal region CA1 than their nonperforated counterparts, which nevertheless outnumber the former by a roughly 9-to-1 ratio (34, 46, 47).These and other circuit-specific differences necessitate a circuit-based approach to understanding the synaptic bases underlying the retention or loss of YA-like memory function in the aging brain. In many ways, the hippocampal system is particularly convenient for such circuit-based approaches (48, 49). Information about the internal and external world is funneled to the parahippocampal system and then relayed via the entorhinal cortex to the dentate gyrus, the first component of the so-called trisynaptic circuit in the hippocampus proper. Granule cells in the dentate gyrus then transmit their computations to hippocampal region CA3 via the mossy fibers, which form very large and anatomically distinct synapses called mossy fiber bouton–thorny excrescence synaptic complexes in the stratum lucidum (SL). CA3 pyramidal neurons then integrate information from their autoassociational connections in the stratum radiatum (SR) and stratum oriens (SO), with both direct entorhinal inputs in stratum lacunosum-moleculare (SLM) and the dentate gyrus inputs in the SL, and convey this information to hippocampal CA1 pyramidal neurons. Neurons in hippocampal CA1 then integrate this information in their basal and apical SR dendrites with direct entorhinal cortical inputs in their most distal, tufted dendrites in the SLM, and represent the first and largest extrahippocampal output from the hippocampus proper. Thus, the computations performed both within individual hippocampal subregions and between them as an interconnected neurocognitive system are complex, and involve a combination of intrinsic (i.e., membrane-bound ion channels that regulate membrane excitability) and synaptic (i.e., ligand-gated ion channels expressed at both excitatory and inhibitory synapses) influences. Additionally, age-related changes at any level of these complex circuits will have downstream consequences on the accuracy/reliability of the information being relayed to extrahippocampal regions via CA1 pyramidal neurons.Given the amount of evidence supporting a possible synaptic explanation for age-related learning and memory impairments in hippocampus-dependent forms of cognition (6–10), we combined patch-clamp physiology, serial section conventional and immunogold electron microscopy (EM), quantitative Western blot analyses, and behavioral characterization using two hippocampus-dependent forms of learning in YA (6- to 8-mo old) and aged rats (28- to 29-mo old) to examine two interconnected hippocampal regions implicated in cognitive aging: Regions CA1 and CA3. We focused on CA1 and CA3 because of their central location in the hippocampal circuit (48–50), their similar laminar dendritic structure (48–50), and their well-documented age-related changes in place field specificity and reliability (51–56).We find that the synaptic architecture and balance of synaptic weights in YA and aged, learning-unimpaired (AU) rats is remarkably similar, but that both are different in aged, learning-impaired (AI) rats. Moreover, this restructuring among “unsuccessful” cognitive agers has an intriguing specificity: It involves only AMPARs, only perforated axospinous synapses, and only hippocampal region CA3, which together shift the balance of synaptic weights that drive action potential output in CA3 pyramidal neurons maladaptively toward an overemphasis of the autoassociational synapses that interconnect CA3 pyramidal neurons.