Formation of ethane and propane via abiotic reductive conversion of acetic acid in hydrothermal sediments

A mechanistic understanding of formation pathways of low-molecular-weight hydrocarbons is relevant for disciplines such as atmospheric chemistry, geology, and astrobiology. The patterns of stable carbon isotopic compositions (δ¹³C) of hydrocarbons are commonly used to distinguish biological, thermogenic, and abiotic sources. Here, we report unusual isotope patterns of nonmethane hydrocarbons in hydrothermally heated sediments of the Guaymas Basin; these nonmethane hydrocarbons are notably ¹³C-enriched relative to sedimentary organic matter and display an isotope pattern that is reversed relative to thermogenic hydrocarbons (i.e., δ¹³C ethane > δ¹³C propane > δ¹³C n-butane > δ¹³C n-pentane). We hypothesized that this pattern results from abiotic reductive conversion of volatile fatty acids, which were isotopically enriched due to prior equilibration of their carboxyl carbon with dissolved inorganic carbon. This hypothesis was tested by hydrous pyrolysis experiments with isotopically labeled substrates at 350 °C and 400 bar that demonstrated 1) the exchange of carboxyl carbon of C₂ to C₅ volatile fatty acids with ¹³C-bicarbonate and 2) the incorporation of ¹³C from ¹³C-2–acetic acid into ethane and propane. Collectively, our results reveal an abiotic formation pathway for nonmethane hydrocarbons, which may be sufficiently active in organic-rich, geothermally heated sediments and petroleum systems to affect isotopic compositions of nonmethane hydrocarbons.

Low–molecular weight (LMW) hydrocarbons (i.e., methane through pentane [C₁ to C₅]) are widespread in marine sediments (1–3) in which they may fuel chemosynthetic ecosystems or zones of intense microbial activity at or below the seafloor. Three principal sources exist for these compounds: biological processes that turn small carbon-bearing compounds into methane (4) and, in smaller quantities, its higher homologs (5), thermal cracking of kerogen and higher hydrocarbons (6), and abiotic production (7). Stable carbon isotopic compositions (δ¹³C) of LMW hydrocarbon are a powerful tool that aids in distinguishing these sources (8, 9). Likewise, carbon isotope patterns within the homologous series of LMW hydrocarbons have diagnostic values (SI Appendix, Fig. S1 A–E). That is, δ¹³C values of thermogenic hydrocarbons, formed from thermal cracking of kerogen, increase with carbon number (e.g., refs. 10 and 11). By contrast, abiotic hydrocarbons formed via Fischer–Tropsch type (FTT) reduction of aqueous CO₂ (12) or from polymerization of methane (9) are increasingly depleted in ¹³C with increasing carbon number, resulting in an inverse isotope trend compared to thermogenic production. Accordingly, methane formed via FTT reactions is typically isotopically enriched in ¹³C (less negative δ¹³C values) relative to thermogenic methane, while nonmethane hydrocarbons formed by polymerization of methane are substantially depleted in ¹³C (more negative δ¹³C values) relative to those formed by FTT reactions and thermogenic processes. Lastly, biogenic methane is generally ¹³C-depleted relative to methane from other sources (4). For ethane and propane, a biological formation pathway involving the reduction of acetate was suggested (5). Volatile fatty acids (VFAs) are produced by thermal decomposition of sedimentary organic matter (13–16) and may accumulate to substantial levels in oil field waters (13, 17) and geothermally heated subsurface sediments (16, 18). Notably, VFAs may serve as potential substrates for the generation of C₂₊ hydrocarbons either via biological processes (5) or via abiotic decomposition involving decarboxylation and/or deformylation (19).In this study, we investigated LMW hydrocarbons in 230 samples of both hydrothermally heated and cold sediments from the Guaymas Basin and observed unusual carbon isotope patterns for ethane, propane, n-butane, and n-pentane. We note that none of the above-described formation pathways can satisfactorily explain the isotopic ordering of the nonmethane hydrocarbons observed in the hydrothermally impacted sediments of the Guaymas Basin (SI Appendix, Fig. S1F). The Guaymas Basin is a unique locality where rapid deposition of organic-rich sediments combined with hot basaltic sill intrusions into the unconsolidated sediments results in rapid heating of young, immature organic matter. This causes the generation of large amounts of complex petroleum-like compounds (20–24), LMW hydrocarbons (25, 26), VFAs (27), and ammonia (28), which migrate upwards with the hydrothermal fluids to fuel a flourishing seafloor ecosystem (29, 30).Given the unusual isotope patterns of C₂ to C₅ hydrocarbons, we explored the potential for an alternative formation pathway involving reductive conversion of VFAs and prior equilibration of their carboxyl groups with ambient dissolved inorganic carbon (DIC). Thus, we conducted sequential hydrous pyrolysis experiments amended with ¹³C-labeled DIC and acetate to assess whether this pathway can produce and explain the observed isotope pattern.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils

Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on ¹³C,¹⁵N-enriched, and fully protonated samples of M₀Aβ_1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and ¹³C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M₀Aβ_1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Amyloid fibrils are highly stable protein deposits found in β-sheet conformations and are notoriously recognized as disruptive agents to cellular function in over 40 human diseases (1, 2). Alzheimer’s disease (AD) is the most pervasive of all known plaque-related diseases and is associated with the presence of amyloid-β (Aβ) peptides in the extracellular space of the brain (3–6). As of 2021, there are ∼6.2 million people in the United States living with Alzheimer’s dementia and ∼50 million worldwide (7), and there is as of yet no cure available for AD. In order to address this epidemic, it is essential that we learn as much as possible about the formation and structure of Aβ plaques, including the detailed features of their catalytic surface, in order to design and develop appropriate treatments to limit the propagation of aggregates and the generation of toxic forms.Aβ is derived from the C-terminal region of the amyloid precursor protein (APP), a membrane protein in neuronal cells, via proteolysis by β- and γ-secretase (8, 9). One of the principal challenges in rationalizing AD etiology is Aβ’s diversity in peptide length, mutations, and posttranslational modifications (10). Their low solubility renders solution NMR ineffective, and high-resolution diffraction analyses have thus far been restricted to shorter peptides with all or most residues being ordered in the fibril core structure (11). Cryogenic electron microscopy (cryo-EM) has made strides in resolution in fibril studies within the past decade (12–18), but faces challenges studying with atomic-level detail due to polymorphism and heterogeneity in the fibril macroassemblies. Studying the individual and collective roles of amyloids at atomic resolution therefore requires alternative, high-resolution, high-throughput techniques for structural analysis. Magic-angle spinning NMR (MAS-NMR) was introduced as a technique with the potential to address these problems (19, 20). Recent technical advances (21, 22) and progress in sample preparation (23) have vastly improved the sensitivity and resolution of the spectra (24). Accordingly, there are now publications describing high-resolution structures of Aβ (25–29) and other amyloid (12, 16, 30–35) fibrils based on distance and torsion angle constraints derived from MAS experiments.To date, all of the known NMR structures of amyloid fibrils were determined using constraints obtained from ¹³C/¹⁵N MAS spectra, which are inhomogeneously broadened and therefore feature well-resolved lines at low spinning frequencies (<25 kHz) (36). However, resolution often remains insufficient for in-depth analysis, and the experiments require relatively large amounts of peptide and extensive signal acquisition periods. Two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP)—promise to circumvent these issues by reducing signal acquisition times or, alternatively, the amount of protein required for the experiment (37). ¹H detection offers a factor of (γ_H/γ_S)^3/2 gain in sensitivity, where S is usually a low γ-spin (38–40) such as ¹³C or ¹⁵N. In these two cases it is possible to achieve a factor of ∼8 or ∼32 gain in sensitivity, respectively. Importantly, ¹H detection also introduces an additional spectral dimension and therefore significantly increases the resolution. In parallel, DNP offers a general approach to enhancing sensitivity by factors of ∼100, dramatically reducing signal acquisition times (by ∼10⁴). It does so by exploiting the high spin polarization of unpaired electrons (of gyromagnetic ratio γ_e ∼660 times larger than γ_H) of a paramagnetic polarizing agent to enhance sensitivity by a theoretical factor of γ_e/γ_H. (41–44) Furthermore, DNP experiments are conducted at ∼100 K, thereby increasing the Boltzmann polarization and sensitivity by another factor of ∼3 over experiments conducted at ambient temperature (45).While these arguments are well established for MAS NMR in many systems, it is less obvious that they are applicable to amyloid samples because spectra of amyloids are known to be broad for a variety of reasons, such as sample purity and polymorphism. Furthermore, ¹H-detected NMR at moderate MAS frequencies (∼20 to 60 kHz) needs to be coupled to different levels of deuteration to ensure high sensitivity and narrow linewidths (42). Accordingly, deuteration with partial reprotonation of the amide or Hα sites has been implemented in pioneering studies on Aβ_1-40 at 20 kHz MAS (46), HET-s(218–289) (47), and D76N-β2m at 60 kHz MAS (48). In addition, selective protonation in Aβ_1-40 fibril methyl groups at 18 kHz MAS has led to highly resolved ¹H-detected ¹³C correlations (49). In deuterated samples, however, the amount of potentially available structural information is significantly reduced, which can impair high-resolution structure determinations. The advent of 0.7 mm MAS rotors that achieve ω_r/2π >110 kHz attenuates ¹H-¹H dipole couplings and allows direct acquisition of multidimensional ¹H data without requiring deuteration (50). Furthermore, the spectra provide assignments and structural information. While a proof-of-concept application of this approach was demonstrated on fully protonated highly regular prion fibrils (51, 52), it is not clear whether this methodology is generally applicable and extendable to the detection of resolved inter- and intramolecular contacts in complex amyloid assemblies.In parallel, our MAS DNP studies on M₀Aβ_1-42 (28, 32, 53) report significant broadening of the NMR lines at cryogenic temperatures, which was attributed to distributions of conformations trapped at low temperature and is therefore inhomogeneous in origin. The loss of resolution associated with the MAS DNP methodology is a major obstacle for the detailed structural study of uniformly labeled amyloid samples. Concurrently, reports of well-resolved spectra at high fields and spinning frequencies suggest that the broadening is homogeneous (54–56). The advent of DNP instrumentation operating at high magnetic fields (18.8 T) and faster MAS (ω_r/2π = 40 kHz) provides an approach to alleviate this limitation by attenuating homogeneous couplings (57). However, this comes at the expense of the enhancement factor, potentially compromising the capacity to carry out expeditious multidimensional and multinuclear correlations. Moreover, NMR spectra of amyloid fibrils are known to suffer from additional debilitating broadening associated with their heterogeneous character (sample purity, polymorphism, etc.), which may mitigate the benefits of high magnetic fields.In this work, we show that high resolution and sensitivity are possible for fibrils of M₀-Aβ_1-42. Notably, we demonstrate rapid resonance assignment and site-resolved detection of numerous site-specific internuclear proximities on submilligram sample quantities via ¹H-detected NMR at ω_r/2π ∼110 kHz and high field (23.4 T/1,000 MHz for ¹H) at room temperature and ¹³C-detected DNP MAS NMR at ω_r/2π = 40 kHz and high field (18.8 T/800 MHz for ¹H) at low temperature. While both ¹H detection and DNP afford increased sensitivity, we estimate, using approaches outlined by Ishii and Tycko (40), that DNP, with our current ε = 22, yields a factor of ∼6.5 higher sensitivity. These results therefore illuminate possible paths for the rapid structure elucidation of amyloid fibrils available in limited quantities.     

α-Synuclein kinetically regulates the nascent fusion pore dynamics

α-Synuclein (α-syn_FL) is central to the pathogenesis of Parkinson’s disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-syn_FL’s role in various membrane fusion steps, they produced conflicting outcomes regarding vesicular secretion. Here, we assess α-syn_FL’s role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-Syn_FL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes influenced α-syn_FL’s inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-syn_FL’s ability to alter fusion pore dynamics. α-Syn_FL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF–resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-syn_FL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca²⁺•syt1 triggered α-syn_FL–resistant SNARE complex formation that rendered α-syn_FL inactive in modulating pore properties. This study reveals a key role of α-syn_FL in controlling vesicular secretion.

Parkinson’s disease (PD) is a protein-misfolding disorder in which nonfunctional α-syn_FL oligomers accumulate within the cell. Several mutations in the gene encoding α-syn_FL are responsible for the dominantly inherited form of PD (1, 2), indicating a crucial role of α-syn_FL in disease pathogenesis. However, the normal cellular function of this intrinsically disordered protein is still unclear.α-Syn_FL is located at the neuronal cell body and presynaptic nerve terminal (3) and is suggested to be involved in synaptic vesicle trafficking (exo- and endocytosis) (4). α-Syn_FL overexpression in cultured hippocampal neurons and coronal slices showed miscellaneous spontaneous and evoked activities (4). Its overexpression, decreased evoking dopamine release from PC12 and chromaffin cells (5). Exocytosis of recycling endosomes was dependent on α-syn_FL’s differential expression in RBL-2H3 mast cells (6). It inhibited α-granule secretion from platelets when stimulated with either ionomycin or thrombin (7). α-Syn_FL single-knockout (KO) mice of two age groups showed no effect in basal synaptic transmission (4); paired-pulse facilitation (PPF) remained unaltered in younger mice but reduced with age (4). α/β/γ-Syn_FL triple-KO (TKO) mice showed elevated basal synaptic transmission in young mice, unlike old mice, which showed a reduction in synaptic transmission (4, 8). PPF remained unaltered in hippocampal and corticostriatal slices of young α/β/γ-syn_FL TKO mice. All these previous studies suggest that α-syn_FL can affect cellular secretion from different cell types.Cellular secretion involves the fusion of secretory vesicles with the plasma membrane in a spatiotemporally coordinated manner. In vitro reconstitution and cell-based studies showed that α-syn_FL interacts with the negatively charged phospholipids and the v-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) synaptobrevin2 (syb2) (9, 10). Ensemble and single-molecule studies demonstrated α-syn_FL’s role in the docking step of membrane fusion (11) by cross-bridging v-SNAREs and acidic phospholipids (12). α-Syn_FL increased clustering of v-SNARE vesicles through its interaction with SNAREs and anionic lipids (13), a mechanism that explains how it engages secretory vesicles close to the active zone of the plasma membrane. It also affected the reclustering of synaptic vesicles (SVs) after endocytosis and thus resulted in reduced neurotransmitter release when overexpressed in hippocampal neurons (14). An in vitro lipid mixing assay also suggested inhibition of vesicle fusion by α-syn_FL’s direct interaction with lipid bilayers (15). α-Syn_FL was involved in priming ATP-induced exocytosis of secretory vesicles in PC12 cells and also affected the late stages of exocytosis (5, 16). Endogenous and overexpressed α-syn_FL was also found to promote fusion pore dilation and cargo discharge, while TKO increased pore closure in neurons and adrenal chromaffin cells (17, 18).Although the above studies demonstrated α-syn_FL’s role in various steps of membrane fusion, they produced conflicting outcomes regarding vesicular secretion. Investigating the direct role of α-syn_FL on membrane fusion steps within the living cell was technically challenging. Furthermore, detailed in vitro studies using a defined system were limited by the time resolution to capture molecular events occurring in physiologically relevant time-scale (micromillisecond). To resolve this issue and to glean insight into α-syn_FL’s mode of action in micromillisecond time-scale, we used a recently described in vitro approach using v-SNARE–reconstituted nanodiscs (NDs) and t-SNARE–reconstituted black lipid membrane (BLM) (19, 20). We investigated whether α-syn_FL regulates vesicular secretion by directly altering the microsecond transitions of individual exocytotic fusion pores (21).During membrane fusion, the fusion pore is the crucial kinetic intermediate through which chemical messengers escape from the lumen of a secretory vesicle into the extracellular space (22). The principal components of exocytotic fusion pores are SNARE proteins (23, 24) and lipids (25). The cytoplasmic domains of v-SNAREs bind to the cognate domains on t-SNAREs, forming trans-SNARE complexes that catalyze the fusion pore formation and serve as the minimal machinery for membrane fusion (26, 27). On a microsecond time-scale, these ephemeral pores either close or dilate as the vesicle membrane fully collapses at the plasma membrane (21, 22, 28). Fusion pores are the site of action of many regulatory proteins that are known to stimulate or limit cellular secretion (29, 30). The ND-BLM system, mentioned above, has revealed that microsecond fusion pore transitions are controlled by the dynamic trans-SNARE complexes and the membrane lipid composition (19, 20). Here, we leverage the strength of this newly developed system to investigate the impact of α-syn_FL on fusion pore properties. Our single-pore measurements are supported by ensemble biochemical studies using v-SNARE NDs and t-SNARE liposomes.α-Syn_FL reduced the open probability of individual pores, without affecting the size of open pores. Open pore denotes the fully open state of a fusion pore unless mentioned otherwise. This observation prompted us to check whether it alters the trans-SNARE assembly. Interestingly, α-syn_FL enhanced trans-SNARE assembly as observed previously (10), and the effect was dependent on the presence of negatively charged phospholipids. Since α-syn_FL binds to both lipids (9, 31–33) and SNAREs (13, 34), we introduced strategies to decouple their contributions in regulating the pore properties. Lipid-binding of α-syn_FL stabilizes its interaction with the trans-SNARE complex and promotes the formation of an inhibitory trans complex. Since the number of trans complexes controls fusion pore properties (19), we investigated whether that impacts α-syn_FL’s functionality. The SNARE copy number directly influences α-syn_FL’s function of modulating fusion pore transitions. Next, we probed α-syn_FL’s pore modulatory capacity in presence of known regulatory factors that affect SNARE complex assembly during membrane fusion (30, 35, 36). α-Syn_FL significantly reduced the pore open probability in presence of Munc13-1 and Munc18, which organize a trans-SNARE assembly to produce an α-SNAP/NSF–resistant complex (37–39). It assists syt1 to act as a membrane fusion clamp in absence of Ca²⁺ (20, 40, 41). Upon Ca²⁺ entry, α-syn_FL no longer elicits its inhibitory action and promotes syt1’s function in dilating exocytotic fusion pores (20).     

Discovery of an ancient MHC category with both class I and class II features

Two classes of major histocompatibility complex (MHC) molecules, MHC class I and class II, play important roles in our immune system, presenting antigens to functionally distinct T lymphocyte populations. However, the origin of this essential MHC class divergence is poorly understood. Here, we discovered a category of MHC molecules (W-category) in the most primitive jawed vertebrates, cartilaginous fish, and also in bony fish and tetrapods. W-category, surprisingly, possesses class II–type α- and β-chain organization together with class I–specific sequence motifs for interdomain binding, and the W-category α2 domain shows unprecedented, phylogenetic similarity with β₂-microglobulin of class I. Based on the results, we propose a model in which the ancestral MHC class I molecule evolved from class II–type W-category. The discovery of the ancient MHC group, W-category, sheds a light on the long-standing critical question of the MHC class divergence and suggests that class II type came first.

The major histocompatibility complex (MHC) class I and class II groups each constitute a multigene family created by gene duplications and subsequent diversifications, with divergent members possessing distinct functions (1, 2). The classical MHC class I and class II molecules play central roles in our immune system by presenting antigens to T lymphocytes (2, 3). Classical MHC class I molecules present peptide antigens to T cell receptors (TCRs) on CD8⁺ T lymphocytes, whereas classical MHC class II molecules present peptide antigens to TCR on CD4⁺ T lymphocytes. After the interaction with the peptide antigen/MHC molecular complex, CD8⁺ T lymphocytes play important roles in the destruction of target cells (e.g., virus-infected cells or tumor cells), while CD4⁺ T lymphocytes play vital roles in helping or regulating antigen-presenting immune cells, including B lymphocytes, which can become antibody-secreting cells (3). Thus, the MHC class divergence is directly linked with our basic immune functions. However, despite decades of MHC research, there has been little progress in understanding the origin of this critical MHC class divergence (4–15).MHC class I and class II genes have been identified not only in bony fish and tetrapods (2, 16) but also in cartilaginous fish, the most primitive jawed vertebrates (17–21). Authentic MHC class I– or class II–like genes have not been demonstrated in the extant jawless fish which possess distinct forms of immune defense. Therefore, the ancestral, antigen-presenting MHC molecule may have arisen, followed by its class diversification, in the common ancestor of jawed vertebrates, in concert with the appearance of their antibody and TCR antigen recognition systems (2).The MHC molecules of the two classes show similarity to each other in their sequences and three-dimensional structures (3). Both classes possess a pair of membrane-distal extracellular domains (peptide-binding domains in the case of the classical MHC molecules) that together form a unique structure composed of an eight-stranded β-sheet topped by two α-helix components and a pair of membrane-proximal extracellular domains that each form an immunoglobulin (Ig)-like, C1-set (22) domain structure. However, the two classes display different combinatorial architectures of these four extracellular domains. A class I molecule is composed of a heavy chain with three extracellular domains (α1 and α2 for the membrane-distal domains; α3 for the membrane-proximal, Ig-like domain) and a noncovalently associated, single, Ig-like domain β₂-microglobulin (β₂-m). In contrast, a class II molecule is composed of two structurally similar chains, α and β, each consisting of two extracellular domains, namely, a membrane-distal domain and a membrane-proximal, Ig-like domain (α1 and α2, respectively, for α-chain; β1 and β2 for β-chain). Furthermore, a class I heavy chain and class II α- and β-chains each possess a connecting peptide (CP)/transmembrane (TM)/cytoplasmic (CY) region. Therefore, a class I molecule has a single CP/TM/CY region while a class II molecule has two.Based on the similarities in the sequences and presumed structures between class I and class II, and on considerations of parsimony, creation of class I from class II was proposed previously (4, 7, 8, 10, 11). From different standpoints, the possible creation of class II from class I was also discussed (6, 9). However, findings of MHC molecules with features which suggest a specific direction of class diversification were not reported thus far (12–15). In the present study, we discovered a category of MHC molecules which possesses dual nature regarding the two MHC classes and, therefore, appears to be critical for the elucidation of the class diversification.     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

From the Cover: Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation

Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer’s disease. A detailed understanding of the underlying mechanistic process of peptide–metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aβ–Zn²⁺ binding features as well as how Zn²⁺ modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn²⁺ effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aβ₄₀ aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn²⁺, the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn²⁺ transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn²⁺ ion, forming a marginally stable, short-lived folded Aβ₄₀ species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aβ₄₀–Zn²⁺ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aβ₄₀.Neurodegenerative disorders, such as Alzheimer’s disease (AD), have their origin in protein misfolding and generation of amyloid aggregates that have been shown to mediate toxic effects on neurons (1, 2). The aggregation of the amyloid β (Aβ) peptide is closely linked to the pathogenesis of AD (3) and is strongly dependent on environmental conditions. Metal ions have been suggested to play a key role in AD pathogenesis (4, 5), and they have been suggested to be involved in generation of amyloid and modulation of cytotoxicity (6, 7). It seems that Zn²⁺ ions have a protective effect on Aβ’s toxicity, at low Zn²⁺ concentrations, whereas higher concentrations may enhance toxicity (8, 9). To this date, it is unclear how the Zn²⁺ levels are altered in the AD brain, and seemingly contradictory studies have reported both elevated and decreased zinc levels (ref. 5 and references therein). In particular, zinc and copper ions have, however, been observed to be enriched in the amyloid plaques from brain tissues of AD patients (i.e., metal ions seem to coaggregate with Aβ) (4, 10, 11).In in vitro studies, Zn²⁺ has been reported to inhibit formation of amyloid fibrils at a metal:peptide ratio of 2:1 (7). However, at high Zn²⁺ concentration it is suggested that amorphous aggregates are formed (12–14). Structural NMR studies on Aβ–Zn²⁺ interactions showed that Zn²⁺ binds to the N terminus of the 40-residue variant of Aβ (Aβ₄₀) (15–17) where the first 16 residues are the minimal peptide sequence for Zn²⁺ binding (16). In Aβ₄₀ the Zn²⁺ ion is coordinated by four ligands, the histidines H6, H13, and H14 and the N-terminal D1 (15). Interaction between Zn²⁺ and Aβ₄₀ causes NMR signal loss of the N-terminal residues (15, 17), and these data suggest that NMR signal loss may be attributed to a chemical exchange process on an NMR intermediate time scale (18) as reported for similar exchanging systems (19–21).In general, modulation of amyloid aggregation by potential inhibitors may occur through interactions with monomers, oligomeric and/or fibrillar species that influence primary and secondary nucleation reactions and/or fibril-end elongation (22, 23). With a kinetic analysis of aggregation profiles the dominating microscopic aggregation mechanism can be determined (24–26). This approach can be applied to characterize which microscopic event(s) during primary and/or secondary pathway is(are) prevented by an inhibitor (23).Despite the huge numbers of studies of the interaction between Aβ₄₀ and metals, and the subsequent effect on self-assembly and aggregation, no detailed model for the molecular mechanism of the modulation of fibril formation has been proposed. Here, we analyze Aβ₄₀ aggregation kinetics in the absence and presence of substoichiometric concentrations of Zn²⁺ ions to elucidate at which level and which microscopic rate constant(s) is(are) modulated by Zn²⁺. In addition, we use NMR spectroscopy to follow the details of the zinc binding and folding of Aβ₄₀ around the zinc ion.     

Natural gas of radiolytic origin: An overlooked component of shale gas

Natural gas is an important fossil energy source that has historically been produced from conventional hydrocarbon reservoirs. It has been interpreted to be of microbial, thermogenic, or, in specific contexts, abiotic origin. Since the beginning of the 21^st century, natural gas has been increasingly produced from unconventional hydrocarbon reservoirs including organic-rich shales. Here, we show, based on a careful interpretation of natural gas samples from numerous unconventional hydrocarbon reservoirs and results from recent irradiation experiments, that there is a previously overlooked source of natural gas that is generated by radiolysis of organic matter in shales. We demonstrate that radiolytic gas containing methane, ethane, and propane constitutes a significant end-member that can account for >25% of natural gas mixtures in major shale gas plays worldwide that have high organic matter and uranium contents. The consideration of radiolytic gas in natural gas mixtures provides alternative explanations for so-called carbon isotope reversals and suggests revised interpretations of some natural gas origins. We submit that considering natural gas of radiolytic origin as an additional component in uranium-bearing shale gas formations will lead to a more accurate determination of the origins of natural gas.

Natural gas is extracted from conventional and unconventional hydrocarbon reservoirs to satisfy current energy demands. Three different origins of natural gas have been distinguished in previous literature including microbial, thermogenic, and abiotic (1). Some researchers also advocate for a low-temperature geocatalytic origin of some natural gases (ref. 2 and references therein). Microbial, thermogenic, and geocatalytic gases are derived from organic matter either by the action of microorganisms or due to elevated temperatures during burial of organic-rich sediments or through geocatalytic generation of nonmicrobial gases at low temperatures. Abiotic processes (3) do not involve organic matter but produce gases through gas–water–mineral interactions in the subsurface by reaction of native H₂ with CO₂ (4). The composition and isotopic signatures of natural gas components are frequently used to determine the origin and maturity of the natural gas (Fig. 1). Natural gases of microbial origin consist mostly of methane that is depleted in ¹³C (δ¹³C ranging between less than −90 and −50‰) (5). In contrast, thermogenic gases from shale gas reservoirs typically contain methane, ethane, propane, and higher n-alkanes with δ¹³C of methane varying between −75 and −20‰ (5) dependent on maturity. Geocatalytic gases mostly consist of methane with δ¹³C between −58 and −41‰ (14). Abiotic gases have a wide range of molecular compositions, and their methane is frequently enriched in ¹³C (δ¹³C ranging between −50 and +10‰) (5).Open in a separate windowFig. 1.Revised Bernard plot after Milkov and Etiope (5). Dark blue line indicates a thermogenic maturation line according to ref. 6 with % R_o increasing from 0.6 to 3. Black dashed lines indicate mixing of radiolytic (R) and thermogenic (T) gas components; the green dashed line indicates the mixing of radiolytic gas with a mixture of primary biogenic gas (e.g., methyl type fermentation) and/or secondary microbial gas (both marked as B in the legend). The brown dashed line indicates mixing of radiogenic (R) and microbial gas derived by CO₂ reduction. CR, CO₂ reduction; F, methyl-type fermentation; SM, secondary microbial; OA, oil-associated (midmature) thermogenic gas; LMT, late mature thermogenic gas after Milkov et al. (1). (Inset) Data points from the Woodford Shale with maturities (7) increasing toward lower dryness values. Data are from radiolytic gases (8) and Barnett and Fayetteville (9), Antrim (10), New Albany (11), Woodford (7), Colorado Group (12), and Alum (13) shales.With the onset of the shale gas revolution early in the 21st century facilitated by horizontal drilling technologies combined with high-volume hydraulic fracturing, natural gas has been increasingly produced in recent years from unconventional hydrocarbon reservoirs such as shales with high organic matter content. Such shales are often associated with high contents of radioactive elements (15–17), and hence, the organic matter they contain is exposed to significant radiation doses over geologic time spans. Naturally occurring radioactive isotopes such as ²³⁸U, ²³⁵U, ²³²Th, ²³⁰Th, and ⁴⁰K and their radioactive daughter products emit α- and β-particles and γ-rays that have penetration depths into the organic matter ranging from <100 µm for α-particles (18) and 1 to 5 mm for β-particles to >50 m for γ-rays (19). Potassium (K) and thorium (Th) are usually associated with detrital minerals. The concentration of radioactive ⁴⁰K is too low in shales to produce significant irradiation of surrounding matter since ⁴⁰K constitutes only 0.012% of all K isotopes (20), while concentrations of radioactive thorium can reach 20 ppm (e.g., refs. 21–23) and uranium (U) up to 2,600 ppm (24). Uranium is typically directly associated with organic matter in black shales (25–28), which will absorb most of the energy released during the U decay. For example, organic carbon in the Alum Shale in Europe with, on average, 100 ppm of U absorbed a 10⁸- to 10⁹-Gy radiation dose over the 500 Ma since its deposition (18, 29, 30).High radiation doses can cause changes in the structure and properties of organic matter (31–35). For the fossil organic matter, kerogen, the most notable changes are an increase of aromaticity, of degree of condensation, and of vitrinite reflectance and a decrease in bitumen content (29, 36–42). Ionizing radiation causes polymerization, cross-linking, dealkylation, and aromatization of organic matter (29, 30, 43, 44) and has been shown to produce short-chain alkanes such as methane, ethane, and propane (8). Additionally, experimental irradiation of organic matter showed the importance of mineral surface area and a presence of clay minerals (44) in disintegration of organic matter and formation of radiolytic products including gases. Furthermore, during irradiation, ⁻H^· radicals form in large quantities (45), which might facilitate radiolytic formation of alkanes.Laboratory-based irradiation experiments (8, 18) with organic matter and crude oils have revealed the formation of radiolytic gas that is mainly composed of H₂ (56 to 96 vol. %), while around 2% of the newly formed gas is composed of methane, ethane, and propane with a linear positive relationship between the radiation dosage and the amount of radiolytic H₂ and alkanes produced (19). These radiolytic hydrocarbons are derived from organic matter but neither through microbial nor through temperature-driven reactions, and they have been found to be depleted in ¹³C (δ¹³C of methane less than −65‰, δ¹³C of ethane less than −45‰, and δ¹³C of propane less than −37‰) (8).We note that previous laboratory-based irradiation experiments using shales and fossil organic matter have not used α-particle irradiation, which mostly occurs in U-rich rocks. Thus, the findings and conclusions presented in this paper are based on the assumption that isotopic signatures of radiolytic gases produced during gamma-ray irradiation in laboratory experiments are equivalent to those resulting from alpha radiation in the geosphere. This is supported by similarities observed between irradiated organic matter in laboratory experiments and in nature. Experiments that used gamma rays from a ⁶⁰Co source (18) demonstrated that irradiated organic matter in shales became slightly enriched in ¹³C requiring that the radiolytic gaseous products are depleted in ¹³C. The slight ¹³C enrichment of irradiated organic matter is also observed in natural U-rich rocks (37, 46–48), and thus, radiolytic hydrocarbons formed in such rocks are also expected to be depleted in ¹³C. This indirectly supports the notion that α-radiation in nature causes formation of ¹³C-depleted radiolytic gases in a very similar fashion to that of gamma radiation in laboratory experiments. However, laboratory data are currently scarce, and future experiments with α-particle irradiation of organic shales as well as controlled temperature parameters within the reaction chamber are needed.This study investigates whether radiolytic methane, ethane, and propane (also referred to as “light alkanes” in the subsequent text) constitute a previously overlooked component of natural gas, especially in organic-rich shale gas plays. We demonstrate that light alkanes derived from the irradiation of kerogen and oil make a nonnegligible contribution to natural gas mixtures from unconventional hydrocarbon reservoirs. By using an isotopic maturation-mixing model on a large set of natural gas data, we quantify the effect of the admixture of light alkanes of radiolytic origin to gases of thermogenic and microbial origin. We also demonstrate that the resulting isotope signatures can lead to misinterpretation of gas origin and maturation levels, and we provide an alternative explanation of the so-called isotope reversals in natural gas from unconventional hydrocarbon reservoirs. We conclude that radiolytic gas derived from organic matter constitutes a previously not recognized type of natural gas that needs to be considered especially in organic-rich unconventional hydrocarbon reservoirs that frequently contain uranium (U) in substantial quantities (15, 16).     

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ₁34.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ₁34.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ₁34.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ₁34.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Herpes simplex viruses (HSV) are human pathogens that switch between lytic and latent infections intermittently (1, 2). This is a lifelong source of infectious viruses (1, 2), in which immediate early proteins drive the onset of HSV replication. Among them, ICP0 enables viral gene expression or reactivation from latency (2–4), which involves chromatin remodeling of the HSV genome, resulting in de novo virus production. In this process, the accessory factor γ₁34.5 of HSV is thought to govern viral protein synthesis (5, 6). It has long been known that γ₁34.5 precludes translation arrest mediated by double-stranded RNA–dependent protein kinase PKR (7–9). The γ₁34.5 protein has also been shown to dampen intracellular nucleic acid sensing, inhibit autophagy, and facilitate virus nuclear egress (10–17). In experimental animal models, wild-type HSV, but not HSV that lacks the γ₁34.5 gene, replicates competently, penetrates from the peripheral tissues to the nervous system and reactivates from latency (18–23). Despite these observations, active HSV replication or reactivation from latency is not readily reconciled by the currently known functions of the γ₁34.5 protein (8–13, 16, 17).Several lines of work demonstrate that tripartite motif (TRIM) proteins regulate innate immune signaling and cell intrinsic resistance to virus infections (24, 25). These host factors typically work as E3 ubiquitin ligases that can synthesize degradative or nondegradative ubiquitination on viral or host proteins. A number of TRIM proteins, for example TRIM5α, TRIM19, TRIM21, TRIM22, and TRIM43, act at different steps of virus replication and subsequently inhibit viral production (26–32). Recent evidence indicates that TRIM23 limits the replication of certain RNA viruses and DNA viruses, including HSV-1 (33). In doing so, TRIM23 recruits TANK-binding kinase 1 (TBK1) to autophagosomes, thus promoting TBK1-mediated phosphorylation and activation of the autophagy receptor p62 and ultimately leading to autophagy. It is unknown whether TRIM23 plays an additional role(s) in HSV infection.Here, we report that ICP0 expression is regulated by the γ₁34.5 protein and TRIM23 during HSV-1 infection. We show that TRIM23 facilitates the proteasomal degradation of ICP0, whereas viral γ₁34.5 maintains steady-state ICP0 expression by preventing K27-linked TRIM23 autoubiquitination that is required for TRIM23 activation. The γ₁34.5 protein also interacts with and stabilizes ICP0, enabling productive infection. Furthermore, we provide evidence that TRIM23 binds to ICP0 and induces its K11-linked polyubiquitination, which triggers K48-linked polyubiquitin-dependent proteasomal degradation of ICP0. These insights establish a model of posttranslational networks in which virus- and host-mediated mechanisms regulate immediate early protein ICP0 stability and thereby lytic HSV replication.     

Serotonin regulates glucose-stimulated insulin secretion from pancreatic β cells during pregnancy

In preparation for the metabolic demands of pregnancy, β cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased β cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a^−/− mice exhibited impaired glucose tolerance despite normally increased β cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in β cells, which increased Ca²⁺ uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the β cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.Pregnancy places unique demands on the metabolism of the mother. As the pregnancy progresses and the nutrient requirements of the fetus increase, rising levels of placental hormones reduce maternal insulin sensitivity, thereby maintaining the maternal/fetal gradient of glucose and the flow of nutrients to the fetus. The mother balances the resulting increase in insulin demand with structural and functional changes in the islets that generate increased and hyperdynamic insulin secretion. β cell numbers increase, the threshold for glucose decreases, and glucose-stimulated insulin secretion (GSIS) increases (1–3). Failure to reach this balance of insulin demand with insulin production results in gestational diabetes (4).However, the changes in the maternal islets are not simply a response to increased insulin demand, as they precede the development of insulin resistance. Instead, these changes correlate more closely with levels of circulating maternal lactogens (prolactin and placental lactogen) that signal through the prolactin receptor on the β cell (5–9). Downstream of the prolactin receptor, multiple pathway components have been identified that contribute to the maternal increase in β cell mass (10–16), but not the changes in GSIS.In response to the lactogen signaling during pregnancy, levels of both isoforms of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin (5-hydroxytryptamine; 5-HT), rise dramatically in the islet (13, 17, 18). Islet serotonin acts in an autocrine/paracrine manner through the Gα_q-coupled serotonin receptor 5-HT2b receptor (Htr2b) to increase β cell proliferation and mass at midgestation and through Gα_i-coupled 5-HT1d receptor (Htr1d) to reduce β cell mass at the end of gestation (13). These dynamic changes in 5-HT receptor (Htr) expression can explain the shifts in β cell proliferation during pregnancy.In addition to Htr2b and Htr1d, β cells also express Htr3a and Htr3b (13). Unlike the 12 other Htr genes in the mouse genome, which encode G-protein coupled serotonin receptors, Htr3a and Htr3b encode subunits of the serotonin-gated cation channel Htr3 (19, 20). Five identical Htr3a subunits or a mixture of Htr3a and Htr3b make up a functional Htr3 channel (21). The channel is predominantly Na⁺- and K⁺-selective, and its opening in response to serotonin actives an inward current and depolarizes the cell membrane (22, 23). Glucose also depolarizes β cells: Rising ATP from glucose catabolism depolarizes the cell by closing ATP-sensitive K⁺ channels, which causes Ca²⁺ to enter the cell through voltage-gated Ca²⁺ channels and trigger insulin granule exocytosis (24).Therefore, we tested the possibility that Htr3 may regulate β cell insulin secretion during pregnancy. We found that lactogen-induced serotonin in the pregnant islet acts through Htr3 to depolarize β cells, thereby lowering the threshold for glucose and enhancing GSIS during pregnancy.     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

Being activated by depolarizing voltages and increases in cytoplasmic Ca²⁺, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.High-conductance voltage- and calcium-activated potassium (BK) channels are homotetrameric proteins of α-subunits encoded by the slo1 gene (1). These channels are expressed in virtually all mammalian tissues, where they detect and integrate membrane voltage and calcium concentration changes dampening the responsiveness of cells when confronted with excitatory stimuli. They are abundant in the CNS and nonneuronal tissues, such as smooth muscle or hair cells. This wide distribution is associated with an outstandingly large functional diversity, in which BK channel activity appears optimally adapted to the particular physiological demands of each cell type (2). On the other hand, small alterations in BK channel function may contribute to the pathophysiology of hypertension, asthma, cancer, epilepsy, diabetes, and other conditions in humans (3–8). Alternative splicing, posttranslational modifications, and regulation by auxiliary proteins have been proposed to contribute to this functional diversity (1, 2, 9–16).The BK channel α-subunit is formed by a single polypeptide of about 1,200 amino acids that contains all of the key structural elements for ion permeation, gating, and modulation by ions and other proteins. Tetramers of α-subunits form functional BK channels. Each subunit has seven hydrophobic transmembrane segments (S0–S6), where the voltage-sensor domain (VSD) and pore domain (PD) reside (2). The N terminus faces the extracellular side of the membrane, whereas the C terminus is intracellular. The latter contains four hydrophobic α-helices (S7–S10) and the main Ca²⁺ binding sites (2). VSDs formed by segments S1–S4 harbor a series of charged residues across the membrane that contributes to voltage sensing (2). Upon membrane depolarization, each VSD undergoes a rearrangement (17) that prompts the opening of a highly K⁺-selective pore formed by the four PDs that come together at the symmetry center of the tetramer.Although BK channel expression is ubiquitous, in most physiological scenarios their functioning is provided by their coassembly with auxiliary proteins, such as β-subunits. This coassembly brings channel activity into the proper cell/tissue context (11, 13). Four different β-subunits have been cloned (β1–β4) (18–24), all of which have been observed to modify BK channel function. Albeit to a different extent, all β-subunits modify the Ca²⁺ sensitivity, voltage dependence, and gating properties of BK channels, hence modifying plasma membrane excitability balance. Regarding auxiliary β-subunits, β1- and β2-subunits increase apparent Ca²⁺ sensitivity and decelerate macroscopic current kinetics (14, 20, 21, 25–30); β2 and β3 induce fast inactivation as well as an instantaneous outward rectification (20, 21, 24, 31, 32); and β4 slows down activation and deactivation kinetics (12, 23) and modifies Ca²⁺ sensitivity (12, 33, 34).It should be kept in mind that β-subunits are potential targets for different molecules that modulate channel function, such as alcohol (35), estrogens (15), hormones (36), and fatty acids (37, 38). Additionally, scorpion toxin affinity in BK channels would tend to increase when β1 is coexpressed with the α-subunit (22).To identify the molecular elements that give β1 the ability to modulate the voltage sensor of BK channels, we constructed chimeric proteins of β1/β2- and β1/β3-subunits by swapping their N and C termini, the transmembrane (TM) segments, and the extracellular loops and recorded their gating currents. Two lysine residues that are unique to the N terminus of β1 were identified to be sufficient for BK voltage-sensor modulation.     

Thermophilization of adult and juvenile tree communities in the northern tropical Andes

Climate change is expected to cause shifts in the composition of tropical montane forests towards increased relative abundances of species whose ranges were previously centered at lower, hotter elevations. To investigate this process of “thermophilization,” we analyzed patterns of compositional change over the last decade using recensus data from a network of 16 adult and juvenile tree plots in the tropical forests of northern Andes Mountains and adjacent lowlands in northwestern Colombia. Analyses show evidence that tree species composition is strongly linked to temperature and that composition is changing directionally through time, potentially in response to climate change and increasing temperatures. Mean rates of thermophilization [thermal migration rate (TMR), °C⋅y⁻¹] across all censuses were 0.011 °C⋅y⁻¹ (95% confidence interval = 0.002–0.022 °C⋅y⁻¹) for adult trees and 0.027 °C⋅y⁻¹ (95% confidence interval = 0.009–0.050 °C⋅y⁻¹) for juvenile trees. The fact that thermophilization is occurring in both the adult and juvenile trees and at rates consistent with concurrent warming supports the hypothesis that the observed compositional changes are part of a long-term process, such as global warming, and are not a response to any single episodic event. The observed changes in composition were driven primarily by patterns of tree mortality, indicating that the changes in composition are mostly via range retractions, rather than range shifts or expansions. These results all indicate that tropical forests are being strongly affected by climate change and suggest that many species will be at elevated risk for extinction as warming continues.Global warming is causing poleward and upward changes in the distributions of many species (1–4). Studies from tropical forests are extremely sparse (1, 5), but the available evidence suggests that many tropical plant species are “migrating” upward (6–8). Accordingly, the composition of tropical montane forests is changing toward increased relative abundances of species whose ranges were previously centered at lower, hotter elevations (6, 8). However, this process of “thermophilization” (9) is generally occurring at velocities slower than concurrent regional temperature increases (6, 8). Furthermore, in at least one tropical site (Volcan Barva, Costa Rica), the observed shifts in tree species composition were mostly a result of increased mortality of species with ranges centered at higher, cooler elevations (8), indicating that species migrations and associated compositional shifts are occurring mostly via range retractions (1). If generalizable, these findings indicate that many tropical tree species have at best a poor capacity to persist under rapidly rising temperatures, suggesting a high risk for species loss in these systems (10).One factor that determines the velocity at which plant species can move through space, and hence migrate, is seed dispersal (11–13). Although most tree species in wet tropical forests are animal-dispersed (14, 15), differences in the relative abundances of species with different dispersal modes could greatly affect the responses of forest communities to climate change. Furthermore, other nonclimatic factors, such as soil and other edaphic factors, can pose significant barriers to species migrations by limiting the movement of species between different habitat types (16–18). For example, the specialization of species on specific soil types or conditions (19, 20) can potentially halt migrations by preventing species from moving into areas with suitable temperatures but unsuitable edaphic conditions (18, 21). Likewise, forest disturbances (anthropogenic or natural) will affect the ability of species to respond to climate change (22, 23). Disturbances can hinder species movements by creating unsuitable conditions and/or by creating dispersal barriers (24). Conversely, disturbances may facilitate range shifts by resetting succession and minimizing priority effects that would otherwise skew competitive interactions, and thereby prevent some species from moving into climatically suitable habitats. Finally, by changing forest structure and cover, disturbances can alter microclimate (25) and influence species and community responses to climate change (9).To date, all studies of tree species migrations from the tropics have looked exclusively at large adult trees (i.e., individuals with diameter at breast height [DBH] ≥ 10 cm). The resultant models of species migrations and community responses to climate change are therefore potentially confounded by the fingerprint of past climatic events or episodic disturbances. The inclusion of smaller size classes (i.e., shrubs and juvenile trees) could potentially help to determine whether observed migrations are responses to episodic or ongoing environmental changes, and hence allow for better predictions of how these forests will fare in the future (26, 27).In this study, we investigated the response of tropical montane forests to anthropogenic climate change in the northern Andes and surrounding lowlands of northwest Colombia (SI Appendix, Fig. S1). This area is poorly represented in ecological studies, and the forest communities have not previously been assessed for climate change responses. To look at forest responses to climate change, we analyzed the patterns of compositional change that have occurred over the last decade in a network of 16 1-ha permanent adult tree census plots with nested subsample plots for censusing the smaller shrubs and juveniles (SI Appendix, Table S1). We assessed the rates of compositional change of both the adult (large individuals) and juvenile (small individuals) tree communities through time. We also tested whether the observed rates of compositional change are related to species’ dispersal modes, plot soil fertility and nutrient concentrations, or forest structural and demographic parameters. Understanding how these factors relate to, and potentially influence, the responses of juvenile and adult tropical tree communities to climate change will enhance our ability to predict and possibly mitigate the effects of future climate change on tropical species, as well as on the valuable services these threatened communities provide for millions of people.