Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly

Degradation of unusual fatty acids through β-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [¹⁴C]acetate and [³H]₂O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [¹⁴C]acetate and [¹⁴C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl–CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl–CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid.Fatty acids (FAs) that accumulate as triacylglycerols (TAGs) in seeds of plants represent a major source of renewable reduced carbon that can be used as food, fuel, or industrial feedstocks. Within the plant kingdom there are greater than 300 different types of “unusual FAs” that contain functional groups (e.g., hydroxy, epoxy, and cyclopropane) or have physical properties useful for replacing petroleum in the chemical industry (1, 2). Unfortunately, most plants which naturally produce these unusual FAs have agronomic features which make them unsuitable as major crops. Over the past 2 decades, most attempts to genetically engineer unusual FAs into oilseed crops or model species have produced only low proportions of the desired FA within TAG (2–5). Additionally, in many cases, accumulation of unusual FAs in transgenic plants is accompanied by a reduction of total seed oil (6–11); in some instances reductions of up to 50% of total seed oil have been reported (7, 10). The endogenous mechanisms that recognize and respond to unusual FAs and result in reduced seed oil accumulation in transgenic plants are unknown. These limited successes and adverse outcomes of oilseed engineering highlight our lack of knowledge on how plants accumulate TAG and indicate a need for better understanding of mechanisms that control seed FA synthesis and accumulation.The net accumulation of a metabolic product is controlled by the combined action of anabolic and catabolic pathways. The FAs that accumulate within TAG are initially synthesized up to 18C and 0–1 double bonds within the plastid. Upon exiting the plastid, newly synthesized FAs may be further modified (desaturated, hydroxylated, etc.) while esterifed to endoplasmic reticulum (ER) membrane lipid phosphatidylcholine (PC) before incorporation into TAG (12, 13). FAs esterifed to glycerolipids have long half-lives (14), with minimal turnover in most tissues (15). A prominent exception takes place in germinating seedlings where TAG is broken down through β-oxidation to produce acetyl–CoA for energy production and gluconeogenesis (16). In preparation for germination, enzymes for TAG degradation accumulate during seed development and lead to a loss of ∼10% of seed oil reserves during late seed maturation (17). Thus, oil levels of mature seeds result from a combination of both FA synthesis and FA catabolism, and an alteration of either process could lead to the reduced oil phenotypes of some transgenic oilseeds.The selective breakdown of unusual FAs within transgenic plants has long been suggested as a major factor limiting production of oilseed crops containing industrial oils (12, 18). Multiple lines of evidence support this hypothesis. The castor (Ricinus communis) FA hydroxylase which produces hydroxylated FAs (HFA) and the California bay medium-chain acyl–acyl carrier protein thioesterase (MCTE) which produces 12:0 (FA nomenclature, # carbons: # double bonds) have been constitutively expressed in tobacco and Brassica napus, respectively (12, 19). In each case, small amounts of the unusual FAs were found in the seeds but not in the leaves. Biochemical analysis of MCTE leaves indicated high MCTE activity and 12:0 production, but no accumulation (19). Thus, the lack of unusual FA accumulation in leaves suggests that unusual FAs are rapidly degraded after synthesis. The accumulation of HFA and 12:0 to significant levels within seed TAG of transgenic plants has been achieved by the use of strong seed-specific promoters (18, 20, 21). Sequestration of unusual FAs in TAG of transgenic plants may limit their adverse effects on membranes and thus allow accumulation in seeds. However, some evidence suggests that unusual FAs are also broken down by β-oxidation in developing seeds. Degradation of HFA through β-oxidation was indicated in the seeds of Arabidopsis plants coexpressing the castor FA hydroxylase along with a bacterial polyhydroxyalkanoate (PHA) synthase, which produces PHA from intermediates of β-oxidation (22). However, it is unclear if the PHA accumulation in the transgenic seeds was due to β-oxidation during the TAG accumulation phase of seed development or during the TAG breakdown phase of late seed maturation. B. napus embryos accumulating 12:0 up to 60% of seed FA had increased levels of a 12:0 specific β-oxidation activity. The substantial rates of β-oxidation during seed development did not reduce total oil accumulation in these seeds, implying that a concomitant increase in FA synthesis compensated for a futile cycle of 12:0 synthesis/degradation (21). Together these results demonstrate that even though seed tissue can accumulate unusual FAs within glycerolipids, the proportion and content of unusual FAs within TAG may still be limited by selective β-oxidation of the unusual FAs.The apparent increased FA synthesis that is proposed to offset unusual FA degradation makes it unclear how production of HFA or epoxy–FA has led to substantially reduced levels of seed oil in multiple transgenic plants (8–11). To better understand the metabolic processes that control TAG accumulation in seeds we investigated the basis of reduced oil accumulation in Arabidopsis plants expressing the castor FA hydroxylase (12). Castor seeds accumulate oil containing ∼90% HFA. However, heterologous expression of the FA hydroxylase in different plants has led to a relatively low proportion of HFA in the oil of transgenic seeds, with the best lines producing ∼17% HFA in TAG (9, 18, 20, 23, 24). The accumulation of HFA within transgenic Arabidopsis seeds is also accompanied by a 30–50% reduction in total seed oil (8–10). Interestingly, endeavors to circumvent the mechanisms limiting the proportion of HFA within TAG through coexpression of the castor FA hydroxylase with HFA-selective TAG-synthesis enzymes increased not only the proportion of HFA in the TAG (to over 25%) but also mostly recovered the seed oil content to that of nontransgenic lines (9, 25). These previous results suggest that inefficient utilization of unusual FAs leads to the reduced seed oil levels. However, it is unknown if the reduced oil content is due to increased catabolism of the unusual FAs through β-oxidation, impairment of FA synthesis, or both.Here we report that production of HFA within the ER of developing Arabidopsis seeds by castor FA hydroxylase expression alone induced a large reduction in FA synthesis and seed oil content, apparently by posttranslational inhibition of plastid localized acetyl–CoA carboxylase (ACCase) activity. Our results indicate that the reduction in FA synthesis is a primary mechanism for the reduced seed oil. Additionally, more efficient incorporation of HFA into TAG by HFA-selective TAG-synthesis enzymes alleviates the inhibition of ACCase activity and increases seed oil content. Thus, we demonstrate in vivo that developing oilseeds can detect inefficient glycerolipid metabolism within the ER and respond by posttranslational down-regulation of de novo FA synthesis within the plastid.     

Talin determines the nanoscale architecture of focal adhesions

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin–talin–actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.Cell adhesion to the ECM is a highly coordinated process that involves ECM-specific recognition by integrin transmembrane receptors, and their aggregation with numerous cytoplasmic proteins into dense supramolecular complexes called focal adhesions (FAs) (1). Actin stress fibers terminate at FAs where actomyosin contractility is transmitted to the ECM, generating traction (2–5). Mechanical tension impinging on each FA is implicated in key steps including the elongation, reinforcement, and maintenance of the FA structures (6). FA mechanotransduction is a major aspect of cellular microenvironment sensing with wide-ranging consequences in physiological and pathological processes (7–10). However, molecular-scale spatial parameters that specify FA nanoscale organization have been difficult to access experimentally. Nevertheless, these are essential to understand how mechanosensitivity arises within such complex molecular machines (11–15).Previously 3D superresolution fluorescence microscopy has unveiled the nanoscale organization of major FA components, whereby a core region of ∼30 nm interposes between the integrin and the actin cytoskeleton along the vertical (z) axis (16). The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins such as talin and vinculin, and a stress fiber interfacial zone that contains actin-associated proteins such as VASP (vasodilator-stimulated protein) and α-actinin. Although such multilaminar architecture signifies a certain degree of compartmentalization within FAs that may serve to spatially constrain protein–protein interactions and dynamics, the structural connectivity, the molecular configuration and geometry of FA proteins, and the molecular basis of their higher-order organization remain unclear.Proteomic and interactome analysis of the integrin adhesome have uncovered several direct and multitier connections between integrins and actin (17–20). This suggests that multiple highly interconnected protein–protein interactions could collectively self-organize into FA structures; such redundancy could also account for the remarkable mechanical robustness of FAs after cellular disruption or perturbation (21). Alternatively, a specific FA component may play a dominant role in regulating FA architecture. Aspects of both scenarios may also act cooperatively or function at distinct stages of FA assembly and maturation. Superresolution microscopy of cells expressing fluorescent protein (FP)-tagged FA components has revealed that talin, a large cytoskeletal adaptor protein, adopts a highly polarized orientation in FAs (16), with the N terminus residing in the membrane-proximal layer and the C terminus elevated by z ∼30 nm to the FA/stress fiber interfacial zone. This led us to hypothesize that an array of integrin–talin–actin linkages may vertically span the FA core, serving a structural role in determining FA architecture (16).To test this hypothesis, we sought to perturb FAs by substituting endogenous talin with recombinant analogs having modified lengths. These were generated by retaining both the N-terminal FERM (band 4.1/Ezrin/Radixin/Moesin) and C-terminal THATCH (Talin/HIP1R/Sla2p Actin-tethering C-terminal Homology, or R13) domains but with selective deletion of the multiple helical bundles within the central region of talin. By using a siRNA-mediated knockdown/rescue approach, we found that such talin analogs were able to support FA formation, clustering of activated integrins, and linkages to the actin cytoskeleton. By mapping the z-position of the FPs tagged at either the N or the C termini, we show that talin and its analogs are linearly extended and oriented in FAs, with their lengths regulating FA nanoscale organization. Chimeric-talin analogs with a 30-nm spacer insertion are also able to support FA assembly, facilitating the precise determination of talin geometry in FAs. Our results indicate that talin is oriented at 15° relative to the plasma membrane, measuring ∼97 nm end to end. FA nanoscale architecture in vinculin-null mouse embryonic fibroblasts (MEFs) retained its stratified organization and talin polarization similar to that in other cell types, suggesting that vinculin is dispensable for the specification of FA architecture. Our measurements demonstrate how the integrin–talin–actin module serves as the primary, and surprisingly modular, structural and tension-bearing core of FAs and geometrically define how such complexes could integrate multiple cellular forces at adhesion sites.     

Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover

Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch.Tissues of the pulmonary, circulatory, musculoskeletal, digestive, and renal systems are subject to mechanical perturbations such as cyclic or continuous stretch. Cells within these tissues are mechanosensitive, responding to these mechanical inputs by changing ion channel configurations (1–3), cytoskeleton organization (4–6), mRNA splicing (7), gene expression (8–10), and posttranslational protein modification (11–13). Although many forms of mechanical stimuli occur physiologically, the most well studied are the responses of cells to cyclic stretch to specifically simulate the contraction/relaxation cycles that occur within the cardiovascular and pulmonary systems (14–17). However, sustained stretch also commonly occurs in tissues—e.g., when injury-induced swelling causes local hydrostatic pressure increase (18), in long-term blood pressure increase (16, 19), during prolonged muscle contraction (20, 21), or when the bladder retains large volume of urine (22, 23). In such situations, tissue stretching may last for minutes or hours without regular relaxation intervals, and likely generates cellular responses that lead to tissue adaptation. However, the cellular responses to sustained stretching are not well studied.Cells exhibit stereotypical morphological responses to stretch. Cyclic uniaxial stretch induces cells to reorient their long axes perpendicular to the direction of stretch. This process is accompanied by a similar reorientation of actomyosin stress fibers and integrin-mediated focal adhesions (FAs) and is thought to minimize tissue resistance to stretch (5, 6, 24, 25). FAs serve as mechanical conduits, transmitting forces generated by stress fibers to the ECM to drive cell movement or ECM remodeling, and also transmitting forces generated in tissues into the cell. The mechanosensitivity of FA assembly and downstream signaling is well documented, and thus they are prime candidates for mediating cellular responses to stretch (26). Indeed, the cytoskeletal reorientation that occurs in response to cyclic stretch requires several proteins that localize to FA, including integrins (27), zyxin (28), paxillin (25), Src, and p130 Crk-associated substrate (p130Cas) (29), and involves sliding reorganization of FAs (6). However, the effects of sustained stretch on FA organization and dynamics have not been explored.Here, we sought to characterize the dynamic response of FAs and cell morphologies to sustained uniaxial stretch. We found FAs to be directionally sensitive to sustained stretching. FA disassembly and loss of cell polarity occurred in cells with their long axes perpendicular to stretch, but not in cells with their long axes parallel to stretch. We show that FA kinase (FAK), Src family kinases (SFKs), and calpain 2 all contribute to FA disassembly and loss of cell polarity, and that FAK is activated specifically in FAs oriented perpendicular to stretch. We hypothesize that putative stress/strain sensing proteins in FAs upstream to FAK and Src align with the long axes of FAs, whereas a majority of FAs aligns with the cellular long axis, resulting in orientation-specific responses. Our findings show that sensitivity of strain/stress orientation at the molecular level propagates up to regulate cell polarity in response to sustained mechanical stimulus.     

ATP allosterically stabilizes integrin-linked kinase for efficient force generation

Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic and intrinsic forces, but how the essential adhesion component integrin-linked kinase (ILK) dynamically responds to mechanical force and what role adenosine triphosphate (ATP) bound to this pseudokinase plays remain elusive. Here, we apply force–probe molecular-dynamics simulations of human ILK:α-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. We identify two key salt-bridge–forming arginines within the allosteric, ATP-dependent force-propagation network of ILK. Disrupting this network by mutation impedes parvin binding, focal adhesion stabilization, force generation, and thus migration. Under tension, ATP shifts the balance from rupture of the complex to protein unfolding, indicating that ATP increases the force threshold required for focal adhesion disassembly. Our study proposes a role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration.

Cells sense and respond to a broad variety of biochemical and mechanical signals from the neighboring cells and the surrounding microenvironment, including the extracellular matrix (ECM). Produced and remodeled by the cells, the ECM serves as a physical scaffold for tissues, but also actively guides tissue development and homeostasis by regulating a broad range of cellular processes, such as adhesion, migration, growth, and differentiation (1, 2). ECM assembly and bidirectional cell–ECM signaling is mediated by the integrin family of cellular surface receptors. Integrin–ECM binding leads to recruitment of filamentous (F-)actin to allow force generation through the contractile actomyosin cytoskeleton. As integrins lack enzymatic activity and do not bind actin directly, their function depends on establishing and maintaining large, multiprotein complexes of actin-binding and -regulatory proteins, the focal adhesions (FAs) (3). FAs are crucial for the precise spatiotemporal coordination of integrin signaling. In addition to sensing ECM composition, integrins and FAs sense mechanical cues and transduce them into biochemical signals through a process termed mechanotransduction (4). These mechanical cues include ECM rigidity, tension, and shear. A key event of mechanosensing is modulation of actomyosin contractility and FA dynamics, determined by the balance of protein association and dissociation. According to current models, collectively termed the molecular clutch model, exposing cells to large, rapidly applied forces or plating cells on stiff substrates leads to large FAs with slower exchange rates of adapter molecules and longer FA lifetimes (5). Thus, precise coordination of the stability of FAs and their actomyosin linkage is required for effective generation of traction stresses during mechanosensing, as well as during directed cell migration (4, 6).A central regulator of FA dynamics and stability downstream of β 1 integrins is integrin-linked kinase (ILK), one of the few essential and evolutionarily conserved components of FAs (7, 8). ILK is the central component of a tripartite IPP complex comprising ILK, PINCH (particularly interesting new cysteine–histidine rich protein), and α-parvin (9). ILK consists of two distinct domains (Fig. 1 A and B): an N-terminal ankyrin-repeat domain, associated with PINCH (10), and a C-terminal atypical kinase domain, which binds to the calponin-homology (CH2) domain of α-parvin (11). The current notion is that the IPP serves as a signal-processing platform by recruiting a variety of proteins. For example, α-parvin directly interacts with paxillin (12–14), PINCH influences receptor tyrosine kinases (15), and both parvin and PINCH bind F-actin (16, 17), connecting the IPP to the cytoskeleton. Although ILK was believed to directly associate with β-integrin tails (18), more recent studies show that ILK rather indirectly contacts integrins by binding to kindlin-2 (19, 20). As kindlin-2 directly interacts with β-integrin (21–25) and binds PIP2 via its PH domain (26, 27), this interaction recruits the IPP to the cell membrane at sites of FAs. While ILK contains a kinase domain with a typical kinase fold, the catalytic function of ILK has been heavily debated (8, 9, 11, 28–31), and ILK is now widely regarded as a pseudokinase. Its obligate interaction partner parvin binds to the putative substrate entry site within the kinase-like domain, and disruption of ILK:parvin binding reduces the localization of ILK to FAs (11). Interestingly, ILK retained its ability to bind adenosine triphosphate (ATP) in the nucleotide-binding cleft in an unusual binding mode (11), and recent work has identified a role for ATP binding in actin stress fiber formation and adhesion morphology (16). However, the molecular mechanism by which ATP impacts ILK functions remains fairly elusive.Open in a separate windowFig. 1.Destabilization of ILK pseudokinase without ATP. (A) Schematic overview of ILK and its associated proteins. (B) The ILK:parvin complex rendered from PDB ID code 3KMW (11). (C) Trajectory median backbone RMSD of ILK(WT) holo and apo and apo ILK(L207W). n = 20 trajectories. **P = 0.001, one-way ANOVA/Tukey honestly significant difference (HSD). (D) PCA of holo and apo ILK(WT) and ILK(L207W). Structures extracted from MD simulations are projected onto PC axes for the first and second PC. Extreme conformations (red and blue) of apo ILK along PC1 and PC2 are overlaid based on a least-squared fit to the C-lobe of the pseudokinase domain, and large-scale motions are indicated by arrows. A schematic of the described motions is presented (see also Movies S1 and S2).The importance of the IPP in regulating the integrin–actin linkage is apparent in deletion studies in mice and cells: ILK depletion results in embryonic lethality caused by failure in epiblast polarization and severe defects in F-actin organization at adhesion sites (32). Furthermore, tissue-specific deletion of ILK leads to heart disease (33), while ILK is also associated with cancer progression (34) and might play a role in aging (35). At the cellular level, ILK deficiency leads to aberrant remodeling of the actin and microtubule cytoskeletons and decreased force generation, which comprises FA formation, cell migration, and ECM remodeling (9, 32, 36–38).While ILK is clearly indispensable for the integrin–actin connection and efficient force generation through integrins, the precise molecular mechanism of force-induced IPP signaling and the role of ATP therein are unknown. One critical determinant of FA kinetics and thus mechanosensing is thought to be force-sensitive changes in protein conformations. These changes depend on the strength and duration of force application: Forces must be transmitted for a period sufficient to induce conformational changes, but large forces also trigger bond breakage or protein dissociation, which will terminate force transmission. Thus, a constant competition between conformational change and bond breakage likely exists at adhesions under high traction stresses (4, 6). Molecular examples include focal adhesion kinase (FAK) and talin: The competition between force-induced unfolding and dissociation from binding partners depends on parameters such as loading rate and direction, and defines their mechanosensing roles (39–44).Regulated by the activity of the chaperone Hsp90, the stability of ILK is critical for the ability of cells to generate traction forces (45). This suggests that force-induced unfolding of ILK could play a role for FA signal transduction by promoting disassembly of force-bearing adhesions (45). To address the mechanosensitivity of ILK and the molecular mechanisms of FA regulation, we employ extensive molecular dynamics (MD) simulations of ILK in equilibrium conditions and under mechanical load coupled to biochemical and cellular experiments. Our studies provide evidence for an allosteric strengthening effect of ATP on the ILK:parvin binding and a role for ATP on the cellular force propagation toward α-parvin. Furthermore, previously unrecognized ILK salt-bridge residues are identified to be critical for ILK:parvin interaction and force transmission. We propose that ILK retained ATP as an obligatory binding partner for structural integrity and efficient force transmission of FAs.     

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.The dynamic connection between the forces generated in the actomyosin cytoskeleton and integrin-mediated focal adhesions (FAs) to the extracellular matrix (ECM) is essential for many physiological processes including cell migration, vascular formation and function, the immune response, and tissue morphogenesis. These diverse functions are mediated by distinct cellular structures including protruding lamellipodia containing nascent FAs that mediate haptotaxis (1), ventral adhesive actin waves that mediate leukocyte transmigration through endothelia (2, 3), and stress fibers (SFs) and FAs that drive fibrillarization of ECM in developing embryos (4, 5). The coordination and interdependence of actin and integrin-based adhesion in these specialized cellular structures are rooted in their biochemical interdependence. Activation of integrins to their high-affinity ECM binding state requires the actin cytoskeleton (6). In turn, integrin engagement with ECM induces signaling that mediates actin polymerization and contractility downstream of Rho GTPases (6, 7). ECM-engaged integrins also affect cytoskeletal organization by physically linking the contractile actomyosin system to extracellular anchorage points (7). Thus, adhesion-associated actin structures are integrated systems that mediate cellular functions requiring coordination of intracellular cytoskeletal forces with ECM binding.Mesenchymal cells generally possess two main types of adhesion-associated actin structures: protruding lamellipodia containing nascent FAs at the cell edge and linear actin bundles in the cell body connected to FAs. Compared with architecturally invariant lamellipodia, adhesion-associated actin bundle structures, including filopodia, the perinuclear actin cap/transmembrane actin-associated nuclear lines, trailing edge bundles, and dorsal SFs, are more diverse in their morphology and less well understood in their architecture and function (8–10). The most-studied actin bundle structure is perhaps dorsal SFs, noncontractile bundles associated at one end with a ventral FA near the cell edge and that extend radially toward the cell center and join with dorsal actin arcs on their other end. How the functional specificity of dorsal SFs is generated apart from the many other distinct adhesion-associated actin bundle structures is not well understood.The functional specificity of adhesion-associated actin structures could be generated either on the adhesion side by compositional differences in FA proteins or on the actin side by differences in the nucleation mechanism and actin binding proteins. On the adhesion side, it is well known that different integrin family members bind distinct types of ECM (11, 12). However, cells adhered to different ECMs all form common structures including lamellipodia, filopodia, and multiple types of SFs. In addition to different integrins, FA function could be regulated by the process of “maturation” in which FAs undergo stereotypical dynamic changes in composition and morphology driven by actomyosin-mediated cellular tension (13, 14). Nascent FAs contain integrins, focal adhesion kinase (FAK), a-actinin, and paxillin (13, 15). When tension is applied, nascent FAs grow and recruit hundreds of proteins, including talin, vinculin, and zyxin (16). These mature FAs then either disassemble or further mature into tensin-containing fibrillar FAs that are responsible for fibronectin fibrillogenesis (17). Thus, the changes in FA size and protein content that accompany FA maturation could give rise to functional specialization of adhesion/actin systems.On the other hand, actin filaments in migrating cells are generated by two main classes of nucleators: the Arp2/3 complex and formins (18). Different nucleating proteins generate different actin organization and geometries, which could in turn dictate functional specificity of adhesions. Arp2/3 forms the branched network in lamellipodia and is thought to be linked to nascent FAs through interaction with FAK (19–21) or vinculin (22). The formin family of actin nucleators, which generates linear actin bundles (23), is more diverse, although formins share a common actin assembly core domain (24), (25). Recent work has begun to ascribe the generation of particular actin structures to some of the 15 formins in mammalian cells, particularly members of the diaphanous family and FHOD1 (26–29). Specifically regarding dorsal SFs, evidence points strongly to polymerization by a formin family member (23, 30–32) but no formin has ever been localized to these SFs or their associated FAs in motile cells. Thus, although formins are clearly critical for forming distinct actin structures, whether they cooperate with FA proteins to specify the function of adhesion-associated actin structures in the cell is unclear.We hypothesized that inverted formin 2 (INF2), found in our recent FA proteome (33), may play a critical role in the formation and functional specificity of adhesion-associated actin structures. INF2 is expressed in cells in two isoforms, one containing a membrane-targeting CAAX-motif that plays a role in mitochondrial fission (34) and a non-CAAX isoform whose function is not well characterized. INF2 is an unusual formin insofar as it contains, in addition to the FH1–FH2 domains that polymerize actin, a WH2-like domain at the C terminus (35) that binds actin monomers to regulate autoinhibition, and also mediates filament severing (35, 36). INF2 also interacts with and inhibits members of the diaphanous family of formin proteins (37). INF2 therefore could have multiple possible roles at FAs in local modulation of actin.Here we explore the role of INF2 in mouse embryonic fibroblasts (MEFs). We find for the first time to our knowledge strong localization of an endogenous formin to FAs at the distal tips of dorsal SFs where it is required for actin polymerization at FAs to form dorsal SFs. We show that INF2 plays a role in controlling morphological, but not compositional maturation of FAs. Strikingly, INF2 is responsible for the formation of one specific class of FAs, the fibrillar FAs that organize the ECM; disruption of INF2 leads to defects in ECM fibrillogenesis. Thus, our study demonstrates that INF2 mediates the formation of dorsal SFs and fibrillar FAs, which together comprise a specific integrated adhesion-associated actin structure responsible for the fibrillogenesis of ECM by fibroblasts.     

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.Ferredoxins (FDXs) are soluble, iron–sulfur proteins that mediate electron transfer in a variety of essential metabolic reactions (1–3) (SI Appendix, Fig. S1). The Chlamydomonas genome encodes 13 FDXs (SI Appendix, Table S1) with localization and redox properties that suggest involvement in specific redox reactions (4). Using a yeast two hybrid approach, a global FDX interaction network was established for Chlamydomonas, suggesting putative roles for FDX1 (originally designated Fd) in redox metabolism, carbohydrate modification, and fatty acid biosynthesis (5); this FDX was already known to function in both linear and cyclic photosynthetic electron flow (4, 6). FDX1 also accepts electrons from FDX–NADP oxidoreductase (FNR) (7) and donates electrons to HYDA hydrogenases (5, 8, 9). Other FDXs may be involved in state transitions, nitrogen metabolism, cellular responses to reactive oxygen species (ROS), and dark anoxia (4, 5, 10–13).The major lipids in thylakoid membranes are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG) (14–17), with MGDG being the most abundant, followed by DGDG (14, 17). In Arabidopsis, three enzyme systems are involved in MGDG and DGDG synthesis (18). In contrast, there is only a single copy each of an MGDG and DGDG synthase gene in Chlamydomonas (19). Chlamydomonas uses the prokaryotic pathway for thylakoid lipid synthesis (20) in which the MGDG species synthesized in chloroplasts contain predominantly C18:3^Δ9,12,15–sn-1 with the unusual hexadeca-4, 7, 10, 13-tetraenoic acid C16:4^Δ4,7,10,13 at the sn-2 position (20). Desaturation of the sn-2 acyl group of Chlamydomonas MGDG requires the CrΔ4FAD desaturase, which is not present in Arabidopsis (21), and MGDG accumulation in Chlamydomonas is reduced in strains that make less CrΔ4FAD (21). Under conditions of nutrient deprivation, there are small amounts of C16:4^Δ4,7,10,13 and C18:3^Δ9,12,15 that are integrated into triacylglycerol (TAG) (22, 23), indicating that fatty acids within TAG can be at least partly recycled from membrane lipids such as MGDG.In this study, we show that a fdx5 mutant (i) cannot grow in the dark despite having a growth rate similar to that of wild-type (WT) cells in the light; (ii) has altered photosynthetic properties, membrane structure, and lipid composition relative to WT cells; and (iii) accumulates higher levels of TAG. In addition, FDX5 appears to interact with the CrΔ4FAD and CrFAD6 desaturases. Together, these results suggest a critical role for FDX5 in fatty acid desaturation and maintaining thylakoid membrane composition and functionality specifically during growth in the dark.     

Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide,the key lipid for skin permeability barrier formation

A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.A skin permeability barrier protects terrestrial animals from water loss from inside the body, penetration of external soluble materials, and infection by pathogenetic organisms. In the stratum corneum, the outermost cell layer of the epidermis, multiple lipid layers (lipid lamellae) play a pivotal function in barrier formation (Fig. S1) (1–3). Impairment of the skin permeability barrier leads to several cutaneous disorders, such as ichthyosis, atopic dermatitis, and infectious diseases.Open in a separate windowFig. S1.Structures of the epidermis, the stratum corneum, acylceramide, and protein-bound ceramide. Acylceramides are produced mainly in the stratum granulosum and partly in the stratum spinosum and are stored in lamellar bodies as glucosylated forms (acyl glucosylceramides). At the interface of the stratum granulosum and stratum corneum, the lamellar bodies fuse with the plasma membrane and release their contents into the extracellular space, where acyl glucosylceramides are converted to acylceramides. Thus, released acylceramides, FAs, and cholesterol form lipid lamellae in the stratum corneum. Some acylceramide is hydrolyzed to ω-hydroxyceramide, followed by covalent binding to corneocyte surface proteins to create corneocyte lipid envelopes. Acylceramide contains ULCFAs with carbon chain lengths of C28–C36. The FA elongase ELOVL1 produces VLCFAs, which are further elongated to ULCFAs by ELOVL4 (29). The ceramide synthase CERS3 creates an amide bond between ULCFA and LCB (17). ω-Hydroxylation of ULCFA is required for acylceramide production. However, the responsible ω-hydroxylase had not been identified previously; its identification is the subject of this research.The major components of the lipid lamellae are ceramide (the sphingolipid backbone), cholesterol, and free fatty acid (FA). In most tissues, ceramide consists of a long-chain base (LCB; usually sphingosine) and an amide-linked FA with a chain length of 16–24 (C16–C24) (4, 5). On the other hand, ceramide species in the epidermis are strikingly unique (Fig. S2A). For example, epidermal ceramides contain specialized LCBs (phytosphingosine and 6-hydroxysphingosine) and/or FAs with α- or ω-hydroxylation (1–3). In addition, substantial amounts of epidermal ceramides have ultra-long-chain FAs (ULCFAs) with chain lengths of 26 or more (≥C26) (4, 5). Unique epidermal ceramides are acylceramides having C28–C36 ULCFAs, which are ω-hydroxylated and esterified with linoleic acid [EOS in Fig. S1; EODS, EOS, EOP, and EOH in Fig. S2A; EOS stands for a combination of an esterified ω-hydroxy FA (EO) and sphingosine (S); DS, dihydrosphingosine; P, phytosphingosine; H, 6-hydroxysphingosine] (1–3, 6, 7). These characteristic molecules may be important to increase the hydrophobicity of lipid lamellae and/or to stabilize the multiple lipid layers. Linoleic acid is one of the essential FAs, and its deficiency causes ichthyosis symptoms resulting from a failure to form normal acylceramide (8). Ichthyosis is a cutaneous disorder accompanied by dry, thickened, and scaly skin; it is caused by a barrier abnormality. In patients who have atopic dermatitis, both total ceramide levels and the chain length of ceramides are decreased, and ceramide composition is altered also (9–11).Open in a separate windowFig. S2.Structure and synthetic pathways of ceramides in mammals. (A) Structure and nomenclature of epidermal ceramides. Epidermal ceramides are classified into 12 classes depending on their differences in the LCB and FA moieties. N-type and A-type ceramides contain C16–C30 FAs (n = 1–15), whereas EO-type ceramides contain C28–C36 FAs (n = 13–21) (6, 7). (B) FA elongation and ceramide synthesis in mammals. The FA elongation pathways of saturated and monounsaturated FAs and the ceramide-synthetic pathways are illustrated. E1–E7 and C1–C6 indicate the ELOVL (ELOVL1–7) and CERS (CERS1–6) isozymes involved in each step, respectively. The differences in the letter size of E1–E7 reflect their enzyme activities in each FA elongation reaction. Cer, ceramide; MUFA, monounsaturated FA; SFA, saturated FA.In addition to its essential function in the formation of lipid lamellae, acylceramide also is important as a precursor of protein-bound ceramide, which functions to connect lipid lamellae and corneocytes (Fig. S1) (12, 13). After the removal of linoleic acid, the exposed ω-hydroxyl group of acylceramide is covalently bound to corneocyte proteins, forming a corneocyte lipid envelope. Acylceramides and protein-bound ceramides are important in epidermal barrier formation, and mutations in the genes involved in their synthesis, including the ceramide synthase CERS3, the 12(R)-lipoxygenase ALOX12B, and the epidermal lipoxygenase-3 ALOXE3, can cause nonsyndromic, autosomal recessive congenital ichthyosis (ARCI) (3, 14–16). CERS3 catalyzes the amide bond formation between an LCB and ULCFA, producing ULC-ceramide, which is the precursor of acylceramide (Fig. S1 and Fig. S2B) (17). ALOX12B and ALOXE3 are required for the formation of protein-bound ceramides (13, 18). Other ARCI genes include the ATP-binding cassette (ABC) transporter ABCA12, the transglutaminase TGM1, NIPAL4 (NIPA-like domain containing 4)/ICHTHYIN, CYP4F22/FLJ39501, LIPN (lipase, family member N), and PNPLA1 (patatin-like phospholipase domain containing 1) (16, 19). The exact functions of NIPAL4, LIPN, and PNPLA1 are currently unclear. Causative genes of syndromic forms of ichthyosis also include a gene required for acylceramide synthesis: the FA elongase ELOVL4, which produces ULCFA-CoAs, the substrate of CERS3 (20).Although acylceramide is essential for the epidermal barrier function, the mechanism behind acylceramide production is still poorly understood, leaving the molecular mechanisms behind epidermal barrier formation unclear. For example, acylceramide production requires ω-hydroxylation of the FA moiety of ceramide. However, the ω-hydroxylase responsible for this reaction was unidentified heretofore (Fig. S1). Here, we identified the cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22), also known as “FLJ39501,” as this missing FA ω-hydroxylase required for acylceramide production. CYP4F22 had been identified as one of the ARCI genes (21), although its function in epidermal barrier formation remained unsolved. Our findings clearly demonstrate a relationship between ARCI pathology, acylceramide levels, and ω-hydroxylase activity.     

MAGUKs are essential,but redundant,in long-term potentiation

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

Discovered 50 y ago, long-term potentiation (LTP) remains the most compelling cellular model for learning and memory. It is generally agreed that NMDA receptor (NMDAR)-dependent LTP is mediated primarily by a postsynaptic modification involving the trafficking of the AMPA-type glutamate receptor (AMPAR) (1–4). During the past decade, effort has been focused on the molecular mechanisms underlying both the constitutive and activity-dependent trafficking of these receptors. A family of synaptic scaffolding proteins, referred to as membrane-associated guanylate kinases (MAGUKs) has featured prominently in these studies (5, 6). These proteins contain three PDZ domains, which are involved in protein–protein interactions. The PSD-95 family of synaptic MAGUKs include PSD-95, PSD-93, and SAP102 and are highly expressed at excitatory synapses (5, 7). In terms of AMPAR basal synaptic trafficking, all MAGUKs appear to play overlapping roles (8). Most research has focused on PSD-95. Overexpressing PSD-95 causes a roughly threefold enhancement in AMPAR excitatory postsynaptic currents (EPSCs) with no change in the NMDAR EPSC (9–13). The enhancement mimics LTP, especially with its selective effect on AMPAR EPSCs (as reviewed in ref. 3). Furthermore, PSD-95 occludes LTP, suggesting a common mechanism (9, 12). This suggests that PSD-95 is an essential step in LTP. However, LTP remains intact in cells lacking PSD-95 (14–16), raising the possibility that PSD-93 or SAP102 may play redundant roles.PSD-95 binds to many synaptic proteins (17, 18) including transsynaptic cell-adhesion proteins (e.g., neuroligins, LRRTMs) (19–24). Of particular interest is ADAM22, a member of a large family of catalytically inactive metalloproteases (25, 26), which, via its binding to the secreted protein LGI1, governs transsynaptic nanoalignment. Deleting either ADAM22 (27) or LGI1 (28) reduces AMPAR synaptic transmission. Critical for ADAM22’s function is the presence of a PDZ binding motif (PBM) at the cytoplasmic C terminus. Thus, expressing a mutated form of ADAM22, which lacks the PBM (ADAM22ΔC5) fails to rescue the defect, resulting from the deletion of ADAM22 (27). Furthermore, AMPAR responses are depressed in ADAM22^ΔC5/ΔC5 knockin (KI) mice (29). Previous results found that the typical enhancement in AMPAR responses seen with the overexpression of PSD-95 or the depression observed with the knockdown (KD) of PSD-95 is absent in LGI1 knockout (KO) mice (27). Interestingly, the depression observed with the KD of SAP102 remained intact (27). Complimentary results are seen with ADAM22^ΔC5/ΔC5 KI mice (29) where overexpression of PSD-95 failed to enhance synaptic transmission. Surprisingly, LTP was intact in these mice. What could account for the dissociation of the enhancing action of PSD-95 and LTP?Here we show an essential role for MAGUKs in both basal synaptic transmission and in LTP. Any one of the MAGUKs can substitute for each other. However, coexpression of the MAGUKs suggests differences in their action. The synaptic enhancement seen with the coexpression of PSD-95 and PSD-93 is no greater than the enhancement observed when singly expressed. In contrast the enhancement seen with the coexpression of PSD-95 and SAP102 is additive. In addition, when MAGUK binding to ADAM22 is eliminated in ADAM22^ΔC5/ΔC5 KI mice, PSD-95 is no longer functional, but the action of SAP102 remains intact. Finally, KD of SAP102 in ADAM22^ΔC5/ΔC5 KI mice abolishes LTP. Based on these results we propose a model in which the AMPAR/TARP/PSD-95 complex binds to ADAM22, a transsynaptic adhesion protein essential for nanocolumn stability, which tethers AMPAR receptors in the nanocolumn. An additional pathway involving SAP102 would hold the AMPAR/TARP/SAP102 complex in the nanocolumn by an ADAM22-independent mechanism.     

Proteo-genetic analysis reveals clear hierarchy of ESX-1 secretion in Mycobacterium marinum

The ESX-1 (ESAT-6-system-1) system and the protein substrates it transports are essential for mycobacterial pathogenesis. The precise ways that ESX-1 substrates contribute to virulence remains unknown. Several known ESX-1 substrates are also required for the secretion of other proteins. We used a proteo-genetic approach to construct high-resolution dependency relationships for the roles of individual ESX-1 substrates in secretion and virulence in Mycobacterium marinum, a pathogen of humans and animals. Characterizing a collection of M. marinum strains with in-frame deletions in each of the known ESX-1 substrate genes and the corresponding complementation strains, we demonstrate that ESX-1 substrates are differentially required for ESX-1 activity and for virulence. Using isobaric-tagged proteomics, we quantified the degree of requirement of each substrate on protein secretion. We conclusively defined distinct contributions of ESX-1 substrates in protein secretion. Our data reveal a hierarchy of ESX-1 substrate secretion, which supports a model for the composition of the extracytoplasmic ESX-1 secretory machinery. Overall, our proteo-genetic analysis demonstrates discrete roles for ESX-1 substrates in ESX-1 function and secretion in M. marinum.

The bacterial pathogen, Mycobacterium tuberculosis, causes the human disease tuberculosis (1). Other pathogenic mycobacterial species cause disease in humans and in animals (2–4). Mycobacterium marinum is an occasional human pathogen that causes disease in ectothermic animals (5, 6). M. tuberculosis and M. marinum use conserved survival strategies during infection of host phagocytic cells (7, 8). Bacteria reside within and then damage the phagosome (9, 10), releasing the bacteria and their products into the cytoplasm (11, 12). Phagosomal damage triggers cytosolic host-response pathways. The released bacterial products dampen the host response to infection. The net result of phagosomal escape is host-cell lysis and bacterial spread (9, 11–13).ESX-1 (ESAT-6 system 1) is a conserved protein secretion system that is essential for phagosomal damage (9, 14) and, consequently, mycobacterial virulence (15–17). Strains lacking an ESX-1 system are retained in the phagosome and attenuated (9, 14). The ESX-1 systems of M. tuberculosis and M. marinum are functionally interchangeable (18). M. tuberculosis ESX-1 substrate genes, regulatory genes, or Region of Difference-1 (RD1) genes complement mutations in the corresponding M. marinum genes (19–21), making M. marinum a popular model for studying the ESX-1 system (22).The ESX-1 machinery includes six conserved proteins (EccA₁, EccB₁, EccCa₁, EccCb₁, EccD₁, EccE₁) that form a complex associated with the cytoplasmic membrane (23, 24). The machinery forms a pore that exports substrates, some of which are processed by the MycP1 protease (23, 25). ESX-1 substrates cross the mycobacterial outer membrane (MOM), localizing to the cell surface and the extracellular environment through an elusive mechanism (26). The majority of ESX-1 substrates are conserved between M. tuberculosis and M. marinum (6, 22), with additional M. marinum–specific substrates (27, 28). ESX-1 substrates include PE/PPE domain proteins of varying size; small, ∼100 amino acid WXG proteins; or glutamine- and alanine-rich proteins (22). PE/PPE proteins and WXG proteins from other ESX systems may contribute to transport across the mycolate outer membrane (29–31).The functions of individual ESX-1 substrates are poorly defined. ESX-1 substrates may serve as regulators that control gene expression within the mycobacterial cell, as components of the secretory apparatus, or as effectors that disrupt membranes or function downstream of lysis (15, 21, 32–36). We have described multifunctional ESX-1 substrates that are regulators and effectors (21).Substrates require each other for export (i.e., they are codependent), posing a major hurdle in understanding substrate function. Initial studies suggested that the deletion of any ESX-1 substrate gene resulted in attenuation in vivo and a complete loss of ESX-1–dependent protein secretion in vitro (16, 37). The number of ESX-1 substrates has since expanded (Fig. 1A) (19, 38–40). Several studies have used transposon-insertion mutagenesis or targeted approaches to disrupt substrate genes and define the role of ESX-1 substrates in secretion and host response (21, 27, 35, 39, 42–44). These approaches have often yielded contradictory results regarding the requirement of substrate genes. Recent studies in M. marinum and M. tuberculosis, including some from our laboratory, have demonstrated that codependent secretion can be uncoupled (21, 35, 38, 39, 43–47). However, a comprehensive quantitative measurement of the requirements of substrates for secretion is lacking.Open in a separate windowFig. 1.ESX-1 substrates are differentially required for EsxA/B secretion and function. (A) The ESX-1 locus and accessory loci. Substrate genes are in pink. The conserved secretory component genes are in teal, including the eccCb₁ gene. Numbers represent M. marinum genome position from Mycobrowser (54). Strains are listed in SI Appendix, Table S1. (B) Hemolytic activity of ESX-1 substrate deletion strains (Left) and complementation strains (Right). Data are the average of at least three biological replicates, in technical triplicate. Dots indicate technical replicates, bars indicate the mean. Error bars are the SD. Statistical significance (sig.) was determined using a one-way ordinary ANOVA (P < 0.001) followed by a Tukey’s multiple comparison test. The hemolytic activity of the ΔeccCb₁ strain was not different from that of the cell-free control (PBS). Significance compared with the ΔeccCb₁ (*), compared with the WT strain (^#). ^#ΔespK/comp, P = 0.0112; ^#ΔespA, P = 0.0247; ^#Δppe68/comp, P = 0.0273; ^##P = 0.0066, ^###ΔespC/comp, P = 0.0047; ^###ΔespJ/comp, P = 0.0032; ^#### and ****P < 0.0001. (C) Macrophage infection of ESX-1 substrate deletion strains (Left) and complementation strains (Right). Data are the average of at least three biological replicates, each in technical triplicate. EthD-1–stained cells were counted from five fields from each well, represented by dots. Statistical significance was determined using a one-way ordinary ANOVA (P < 0.0001) followed by a Tukey’s multiple comparison test. Significance compared to the ΔeccCb₁ (*), compared to the WT strain (^#). The cytolytic activity of the ΔeccCb₁ strain was significantly different from that of the cell-free control (***P = 0.0009). **** or ^####P < 0.0001, **P = 0.0053, ^##P = 0.0043. A summary of hemolytic and cytolytic activity is given in SI Appendix, Table S4. (D) Western blot analysis measuring protein secretion. Protein (10 μg) was loaded in each lane. Western blots are representative of at least three biological replicates. OD, optical density.We used a proteo-genetic approach (i.e., using genetics to inform the proteome) to define the specific contribution of each known ESX-1 substrate to protein secretion in M. marinum (48). We generated 12 M. marinum strains with unmarked deletions of individual ESX-1 substrate genes and the 12 corresponding complementation strains. We measured ESX-1 function in each M. marinum strain, demonstrating separable contributions by ESX-1 substrates to the lytic and cytolytic activity of M. marinum. Our studies identified M. marinum proteins with secretion profiles similar to ESX-1 substrates, and potential new ESX-1 substrates. We provide a model for the spatial order and transport of ESX-1 proteins across the MOM that is consistent with previous protein localization and interaction studies. Overall, our studies provide insight into the separable role of ESX-1 substrates in protein secretion and virulence.     

Meteorite-catalyzed syntheses of nucleosides and of other prebiotic compounds from formamide under proton irradiation

Liquid formamide has been irradiated by high-energy proton beams in the presence of powdered meteorites, and the products of the catalyzed resulting syntheses were analyzed by mass spectrometry. Relative to the controls (no radiation, or no formamide, or no catalyst), an extremely rich, variegate, and prebiotically relevant panel of compounds was observed. The meteorites tested were representative of the four major classes: iron, stony iron, chondrites, and achondrites. The products obtained were amino acids, carboxylic acids, nucleobases, sugars, and, most notably, four nucleosides: cytidine, uridine, adenosine, and thymidine. In accordance with theoretical studies, the detection of HCN oligomers suggests the occurrence of mechanisms based on the generation of radical cyanide species (CN·) for the synthesis of nucleobases. Given that many of the compounds obtained are key components of extant organisms, these observations contribute to outline plausible exogenous high-energy–based prebiotic scenarios and their possible boundary conditions, as discussed.Hypothesizing formamide (FA) as parent molecule, we explored its potentiality in synthetic processes when exposed to proton irradiation. The purpose of this analysis is to verify a possible prebiotic scenario consisting of FA, considered here as starting one-carbon atom material, of proton beams mimicking solar energetic particles as energy source, and of meteorites as catalysts. The rationale of this approach is that the results could help in outlining exogenous prebiotic models and their boundaries.FA (NH₂CHO) is becoming one of the most intensively studied precursors for prebiotic syntheses of compounds potentially relevant for the origin of life (1–4). FA is a ubiquitous molecule in the universe. It has been detected in galactic centers (Sgr A and Sgr B), in star-forming regions of dense molecular clouds, in high-mass young stellar objects, in the interstellar medium and in comets and satellites (5–14).With the appropriate mineral as catalyst, different ensembles of intermediates of genetic and metabolic apparatuses are simultaneously synthesized from FA under thermal conditions (i.e., by heating liquid FA between 333 and 453 K at room pressure). DNA and RNA components (15–21), amino acids (22, 23), sugars (24), and carboxylic acids (25, 26) have been obtained. Minerals tune the selectivity of these transformations (1, 2), the mechanistic pathways for the synthesis of nucleobases requiring pyrimidine (27–30) or imidazole intermediates (31, 32).Within the solar system, ionizing cosmic radiation is generated by the Sun [solar cosmic rays (SCRs), primarily protons accelerated by flares and coronal mass ejections to energies typically of tens to hundreds megaelectronvolts] and is also formed by particles coming from the deep universe [galactic cosmic rays (GCRs)] (33). The SCRs and GCRs differ in their components and energy spectra, but their overwhelming component is protons.As an alternative to heat, radiation is a plausible energy source for prebiotic processes (34–36). After irradiation, FA can reach singlet (S₁ and S₂) or triplet (T₁) excited states by n(HOMO)/π*(LUMO) and π(HOMO-1)/π*(LUMO) electronic transitions, respectively (37). The energy profiles for the decomposition of excited states of FA have been studied at different theoretical levels, suggesting the formation of reactive nitrogen- and carbon-centered radical species (38–41). The synthesis of low–molecular-weight compounds (such as carbon oxides COx, HCN, isocyanide, and NH₃) occurs by fast quenching or coupling reactions followed by dehydration, elimination, and dehydrogenation processes. This was shown by a variety of high-energy condensed and gas-phase processes, consisting on UV photodecomposition (42, 43), laser spark (44), synchrotron light (45), low-energy protons (200 keV) (46), and Lyman-α photons (47) irradiations. On the other hand, radiative conditions supposedly exist under which the radical species might react to yield complex and biologically relevant organic compounds. As an example, the synthesis of purine nucleobases by energetically favorable multistep addition of cyanide radicals (•CN) on FA has been predicted on the basis of the density function theory (B3LYP with the 6–311 G d.p basis set) (48). The CN• radical was discovered in interstellar space and in envelopes of giant stars (49). In addition, the energy profile of excited states of FA are profoundly modified after interaction with metal ions (50). The interaction of FA with Li⁺ and Na⁺ leads to a pronounced shift of the n-π* state to higher energies (while the π-π* state moves in opposite direction) with strong geometrical effects favoring the formation of •CN radicals. Thus, the interaction of FA with metal ions or minerals may generate conditions that are energetically favorable to the increase of the structural complexity of end products (51).Meteorites might have played a major role in the chemical inventory of the early solar system for origin-of-life processes, behaving (52, 53) as carriers of organic molecules in connection with the Earth heavy-bombardment period (54, 55) and preceding the supposed earliest existence of living organisms (56). Evidence of the catalytic properties of meteorites in prebiotic chemistry was recently provided by thermal processes of condensed-phase FA at relatively high temperatures (333 and 413 K) (57, 58) or at the most extreme temperature conditions modeling impact events in Earth’s atmosphere or surface (3, 59). During their wandering, meteorites are exposed, under an extreme variety of temperature conditions, to ionizing radiation of cosmic origin (60) able to excite and dissociate FA (the ionization energy of the FA being ∼11–15 eV).Thus, we asked the question: May protons and meteorites be the benign environment for the formation of biomolecules from FA? As a result, we observed an unprecedented one-pot synthesis of nucleosides, nucleobases, and other intermediates of the genetic and metabolic apparatuses by 170-MeV proton irradiation of FA. The formation of nucleosides is particularly noteworthy because of the known difficulty of obtaining these key components of nucleic acids in prebiotic conditions.     

Effects of geometry and chemistry on hydrophobic solvation

Inserting an uncharged van der Waals (vdw) cavity into water disrupts the distribution of water and creates attractive dispersion interactions between the solvent and solute. This free-energy change is the hydrophobic solvation energy (ΔG_vdw). Frequently, it is assumed to be linear in the solvent-accessible surface area, with a positive surface tension (γ) that is independent of the properties of the molecule. However, we found that γ for a set of alkanes differed from that for four configurations of decaalanine, and γ = −5 was negative for the decaalanines. These findings conflict with the notion that ΔG_vdw favors smaller A. We broke ΔG_vdw into the free energy required to exclude water from the vdw cavity (ΔG_rep) and the free energy of forming the attractive interactions between the solute and solvent (ΔG_att) and found that γ < 0 for the decaalanines because −γ_att > γ_rep and γ_att < 0. Additionally, γ_att and γ_rep for the alkanes differed from those for the decaalanines, implying that none of ΔG_att, ΔG_rep, and ΔG_vdw can be computed with a constant surface tension. We also showed that ΔG_att could not be computed from either the initial or final water distributions, implying that this quantity is more difficult to compute than is sometimes assumed. Finally, we showed that each atom’s contribution to γ_rep depended on multibody interactions with its surrounding atoms, implying that these contributions are not additive. These findings call into question some hydrophobic models.Many techniques in computational biophysics require the computation of free-energy differences. However, directly computing these free-energy differences with quantum or classical molecular dynamics (MD) methods has proven challenging because doing so requires long simulation times. To make such computations more tractable, techniques have been developed based on the observation that these free-energy changes could be computed by combining estimates of the changes in vacuum, where they are usually easier to compute, with estimates of the solvation energies (ΔG) of the initial and final states (1, 2).One common technique for computing ΔG is to break it into electrostatic (ΔG_el) and hydrophobic (ΔG_vdw) terms (3, 4). In this breakdown, ΔG_vdw is the free energy required to place an uncharged van der Waals (vdw) cavity into solution, and ΔG_el is the free energy of charging the cavity once it has been placed into solution. Several techniques, including the Poisson–Boltzmann equation (5), the generalized Born model (6), structured continuum approaches (7–9), and integral equation methods (10), have been developed to compute ΔG_el. In the present study, we continue a study of ΔG_vdw begun previously (11–13).Many models have been constructed to compute ΔG_vdw. The first of these models, and perhaps the most widespread, isΔG_vdw = γ_vdwA + b, [1]where γ_vdw is a positive constant independent of the properties of the molecule and b is the energy of solvating a point-like solute (2, 14–17). This model follows from assuming that ΔG_vdw for microscopic cavities should scale with A in the same manner as for macroscopic cavities (18). Alternatively, several studies have pointed out that experimental measurements often are not consistent with Eq. 1 (19–27). These studies have argued that ΔG_vdw should be split into purely repulsive (ΔG_rep) and attractive (ΔG_att) parts:ΔG_vdw = ΔG_rep + ΔG_att.[2]Often, this breakdown follows that of Weeks et al. (19) and Chandler et al. (20). These studies usually then proceed to assume that ΔG_rep is a linear function of A or the molecular volume (V),ΔG_rep = γ_repA + b, ?or[3]ΔG_rep = ρ_repV + b, ?or[4]ΔG_rep = γ_repA + ρ_repV + b, [5]where γ_rep and ρ_rep are positive constants independent of the properties of the molecule. Studies (28–31) have argued that ΔG_rep should obey Eq. 3 for large solute molecules, Eq. 4 for small solutes, and Eq. 5 in a cross-over regime. Alternatively, one could construct other functional forms that would approach Eqs. 3 and 4 in the appropriate limits, such asΔG_rep = 1/((1/γ_repA) + (1/ρ_repV)).[6]Some researchers (23, 24, 27) have then estimated ΔG_att by integrating over approximate solvent densities:ΔGatt=〈Uatt(1)〉ρ¯,[7]where this average was taken over ρ¯, an approximate solvent distribution, and U_att was the attractive dispersive interaction energy between the solute and solvent, as described in Materials and Methods.In three previous studies, we obtained computational results that appeared to contradict some common expectations of ΔG_vdw (11–13). For example, the idea that ΔG_vdw drives the initial collapse during protein folding by opposing the formation of cavities in the solvent with an energy penalty that increases with A has frequently been discussed (2, 32, 33). However, in the first of our studies (11), where we computed ΔG_vdw for a series of glycine peptides ranging in length from 1 to 5 monomers, we found that although ΔG_vdw was linear in the number of monomers, as would be expected if it scaled with A, ΔG_vdw was negative and decreased with the number of monomers, seemingly implying that γ_vdw was negative. In the second of our studies (12), we computed ΔG_vdw for a set of decaalanine conformations and found that ΔG_vdw was negative for these peptides as well. Finally, in the third study (13), we computed ΔG_vdw for a series of alanine peptides ranging in length from 1 to 10 monomers, with the peptides either held in fixed, extended conformations or allowed to assume a normal distribution of conformations. We once again found that ΔG_vdw was negative, and it decreased with the number of monomers when the peptides were held in extended conformations but increased with the number of monomers when the peptides were allowed to adopt a normal distribution of conformations.To better understand these findings, here we computed with free-energy perturbation (FEP) (34, 35) not just ΔG_vdw but also ΔG_rep and ΔG_att for four conformations of decaalanine from a previous study (12) and a series of alkanes that has been examined in the literature (22). As discussed below, none of Eqs. 1 and 3–7 are consistent with our data. Each atom’s contributions to ΔG_rep, ΔG_att, and ΔG_vdw appear to depend on their chemical surroundings and the geometry of the molecular surface. We do not know of a theory that can explain the calculations presented here.     

A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HA_PR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI⁺), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HA_PR8. This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HA_PR8. Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Influenza remains a global health threat. Seasonal strains of influenza A and B cause an estimated 5 million cases of severe infections and 500,000 deaths per year (1). Influenza pandemics have caused even greater morbidity and mortality. During the H1N1 pandemic of 1918 to 1919, 500 million people, approximately one-third of the world’s population at that time, were estimated to have been infected with this strain, leading to 50 million deaths (2). The H1N1 pandemic of 2009 is estimated to have caused up to 575,000 deaths (2). Currently, three types of influenza vaccines are offered annually in the United States: a recombinant virus expressing influenza proteins, chemically inactivated virus, and live attenuated virus (3). Regardless of the vaccine type, multiple strains are included to increase the chances of developing sufficient protection against major circulating influenza strains. However, these vaccines primarily elicit a neutralizing antibody response that is sensitive to changes in the influenza virus due to antigenic drift and shift (4). Antigenic drift results from an accumulation of random mutations in influenza antigens, like hemagglutinin (HA), altering sites recognized by the immune system (4). Influenza A strains can also undergo antigenic shift, whereby two different influenza strains infect the same cell to form a reassortant virus with new antigenic properties (4). Due to limited immunity in the population, these new strains are highly virulent, causing widespread epidemics and disease (4). With antigenic drift and shift, vaccine-mediated protection against circulating strains has been insufficient (5). Influenza vaccines that elicit more robust and long-term protection are therefore needed. Notably, if an influenza vaccine with ≥75% efficacy were to be broadly used in the United States, an estimated 19,500 deaths a year could be prevented and direct healthcare costs reduced by $3.5 billion (6).For many years, efforts to improve influenza vaccines have focused on eliciting an immune response for full, broad protection against both circulating and future strains of the virus. These studies have shown that, in general, neutralizing antibodies are sufficient for homologous protection (7). However, achieving heterologous protection may require more broadly neutralizing antibodies or nonneutralizing antibodies able to activate effector immune cells (5). Previous studies have found that passively transferred nonneutralizing monoclonal antibodies can be potently protective in a mouse influenza challenge model (8–10). Several novel strategies have attempted to generate a nonneutralizing response against influenza. For example, vaccines have been created to specifically target the conserved stem region of HA (11–13).Nonneutralizing antibodies stimulate effector cell mechanisms, including antibody-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), both of which require activation of the Fcγ receptors (FcγRs) (14). Specific isotypes of IgG antibodies are associated with FcγR modulation and subsequent ADCC activation, including the IgG1 and IgG3 subtypes in humans, as well as IgG2a and IgG2c subtypes in mice (15–19). IgG2a and IgG2c isotypes are functionally equivalent and mouse strain-dependent, with IgG2c present in C57BL/6J mice (20). Recent studies have demonstrated that natural infection by influenza and vaccination elicit nonneutralizing antibodies with effector functions that contribute to protection (5, 9, 21–27). In mouse and nonhuman primate challenge models, ADCC-mediating antibodies have demonstrated protection against both homologous and heterologous influenza challenge (9, 28).Recently, we developed a single-cycle herpes simplex virus (HSV) vaccine that completely protects against vaginal, skin, and ocular challenges by HSV-1 and HSV-2 (29, 30). Protection elicited by this vaccine, designated ΔgD-2 for its lack of the essential glycoprotein D (gD) gene, is transferable via passive infusion of immune sera to naïve wild-type mice but not to mice lacking the Fcγ common chain (30). The immune response elicited by ΔgD-2 primarily elicits nonneutralizing antibodies with high levels of FcγRIV-activating function.We asked whether ΔgD-2 could be used as a vaccine platform to induce broadly protective FcγRIV-activating antibodies against a heterologous antigen, such as influenza HA. In this study, we demonstrate that our recombinant vaccine, ΔgD-2::HA_PR8, elicits protection against influenza with a high proportion of FcγRIV-activating antibodies. Additionally, anticipating the use of ΔgD-2 as a vaccine vector against other pathogens, we tested whether our construct would still be protective in mice lacking interferon (IFN) function. Many humans have inborn errors in their IFN signaling pathways, leading to more lethal outcomes in infection (31). Patients with such deficiencies are disproportionately represented among HSV encephalitis cases and are often diagnosed only after presenting with serious symptoms (32–38). This at-risk population underscores the importance of eliciting protection against HSV in the absence of a functional IFN-α/β response. Additionally, many pathogens, such as dengue virus, require mouse models lacking IFN function, and for ease of testing, an efficacious vaccine should remain functional in these mice (39–41). In this study, we demonstrate that ΔgD-2 is a versatile, immunogenic vaccine vector that provides a strong FcγRIV-activating immune response against heterologous pathogens, while maintaining its protective benefit against HSV, in both wild-type and IFN-deficient mice.     

Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau

Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255–441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50–70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth.The question of whether there is a unified mechanism for amyloid formation that contributes to the progression of neurodegenerative diseases, including Alzheimer’s disease (AD), is a subject of intense debate (1). This view is fueled by the observation that a wide range of amyloidogenic proteins or peptides with different primary sequences can ultimately aggregate into highly structured amyloid fibrils with similar morphologies (2). It has been recognized that a common feature of neurodegenerative diseases is associated with the accumulation and deposits of misfolded proteins that affect various cell signaling processes (3). Among them, the pathological form of a microtubule-associated protein, tau, can dissociate from microtubules and aggregate, resulting in the deposition of insoluble neurofibrillary tangles in neuronal cells (3, 4). Several lines of evidence suggest that small aggregated protein intermediates, that may constitute soluble oligomers and precede the formation of highly structured fibrils, are primary toxic species contributing to the early onset of neurodegenerative diseases (4–6). For example, the presence of granular tau oligomers with prefilamentous structures in transgenic mice was found to correlate with the earliest sign of cognitive decline and memory loss (6). Although early aggregation intermediates formed before fibrils may represent effective targets for common therapeutic intervention, knowledge about their structure and properties is only now emerging. The studies of early intermediates—many of which currently focus on amyloid-β, and comparably few on tau oligomers—are challenging, given the transient nature and complex equilibria involving monomer and oligomer species (4, 7, 8). Recently, several computational studies have identified structural and dynamic features of intermediate aggregates at the molecular level (9, 10), as well as possible driving forces at different aggregation stages (10, 11). Characterization of freeze-trapped amylospheroids by solid-state NMR showed that intermediate structures of amyloid-β aggregates are composed of single conformers containing parallel β-sheets (12). Still, direct experimental observations of transient aggregation and/or folding intermediates remain sparse (5, 12–15).We have previously established a broadly applicable spectroscopic method, Overhauser dynamic nuclear polarization-enhanced NMR relaxometry (ODNP) (16, 17), to probe changes in translational diffusivity of local water within 1 nm of nitroxide radical-based electron spin labels tethered to specific protein residues, and successfully reported on the study of protein folding, protein aggregation, and conformational changes of globular and membrane protein segments (18–21). ODNP has revealed increased heterogeneity, i.e., dispersion, in local water diffusivity on the surface of a folded protein, compared with its unfolded counterpart or folding intermediate (20). The basic connection between surface water diffusion and protein structural transformation is that conformational changes, locally or globally, are modulated by the shift in balance between protein surface−water versus protein−protein interactions. The varying stability of this local hydration shell will be collectively reflected in varying retardation of water diffusivity within about 1 nm of the protein surface of interest (22, 23). It has been reported that this surface water dynamics not only varies from surface to surface but is also dramatically heterogeneous from region to region on a given protein surface (20, 22). Terahertz measurements have shown that structural rearrangements of a protein at different folded states exhibit different Terahertz absorption energies that were attributed to changes in the coupling dynamics between the protein surface and hydration water (23). However, the heterogeneous water dynamics landscape of a structurally evolving or interacting protein in situ and in dilute solution has been elusive to probe to date. This study, aided by ODNP, sets out to exactly do that, namely, to probe the structural transition of tau in the early stages of aggregation in solution.Tau monomers belong to a family of intrinsically disordered proteins (IDPs) that, upon misfolding and aggregation, form highly structured amyloid fibrils (24). In this work, we studied the tau variant truncated between residues 255 and 441 (also known as Δtau187, Fig. 1A) of the longest isoform of the human tau protein (25) that includes all four microtubule-binding repeat domains (MTBD) and the C-terminal region (25, 26) (Fig. 1A). This and related truncated variants of tau have been shown to have greatly accelerated aggregation kinetics to form mature fibrils after approximately >12 h of aggregation (24–26). The known hydrophobic hexapeptide stretches, ²⁷⁵VQIINK²⁸⁰ (PHF6*) and ³⁰⁶VQIVYK³¹¹ (PHF6), found at the beginning of the second and third repeat segment are highly amyloidogenic, and form core regions of the tau fibrils (27, 28) with propensities for parallel in-register β-sheet structures reported (28–30). To induce tau fibrillization in vitro, polyanions, such as heparin or arachidonic acid micelles, are typically added to the solution (31). Although the molecular basis for this process is not fully understood, the structure of tau fibrils induced by heparin in vitro displays similar propensities to those found in AD patient tissues (31). Furthermore, heparin ultimately does not remain in the mature fibril core (32). Thus, heparin-induced tau aggregation has served as an accepted model for pathological tau fibrillization (30–32).Open in a separate windowFig. 1.Visualization of Δtau187 aggregates at different aggregation time (t). (A) Δtau187 consists of four microtubule-binding domains indicated as 1–4, where the amyloidogenic hexapeptide region of the third repeat is marked as a black box. The positions of the spin labels at mutated cysteine residues are indicated. (B) Cryo-TEM micrographs display the morphologies of Δtau187 (322C) aggregates after various aggregation times. (Scale bar, 100 nm.) (C) Cross-linked Δtau187 (322C) aggregates at different aggregation time analyzed by SDS/PAGE. (D) A schematic representation of the hypothesized Δtau187 species evolving at five aggregation stages. (E) Aggregation time dependence of τ of hydration water near different spin-labeled residues of Δtau187.In this study, we focused on capturing the structural transformation of the heparin-induced aggregation of Δtau187 in situ under ambient solution conditions and as a function of aggregation time. To achieve this, we used ODNP to probe the translational diffusion dynamics of surface water on protein surfaces and concurrently continuous wave (cw) electron spin resonance (ESR) line shape analysis (33, 34) to monitor protein side-chain mobility and interprotein contacts using the same protein sample (16, 17). Both methods rely on the site-directed mutagenesis and spin-labeling of the protein at a single-cysteine site with an (1-oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) methyl methanethiosulfonate (MTSL)-based nitroxide label, whose side chain is commonly termed “R1” (35). Δtau187 was singly R1 spin-labeled across several key residues of its third repeat MTBD and C terminus. The ODNP measurement was used to derive (as discussed in SI Overview of the ODNP Technique in detail) the translational correlation time, τ, of water within ∼1 nm of the R1 label tethered to the protein surface, together with two relaxivity parameters termed “k_σ” and “k_ρ” that report on contributions from freely diffusing water near the protein surface and bound water to the protein surface on the timescale of a few nanoseconds or longer, respectively. Thus, when a characteristic dispersion, i.e., an increased heterogeneity, of surface water diffusivity develops on the Δtau187 surface, as it transforms from monomeric species to oligomeric and larger fibrillar species, this indirectly shows that Δtau187 is undergoing structural transformation (18). However, ODNP analysis alone cannot differentiate between intraprotein or interprotein structural changes, nor does it offer population information. The cw ESR concurrently measures the structural and dynamic features of the same R1 spin-labeled protein species, as the backbone rearranges and interprotein contacts form during protein assembles. Specifically, we carried out quantitative ESR line shape simulation to determine the spin label mobility, and the population (percent) of the spin label in the respective motional states, and as embedded in parallel β-sheet arrangements. The spin label may experience distinct motional environments—slow vs. fast—that can be attributed to a local protein environment in which R1 is embedded at an intertau interface vs. one in which R1 is freely rotating in the solvent. Their evolving populations with increased aggregation time can be quantified. If R1 of Δtau187 at a specific position gets embedded in β-sheet structures, the evolving content of the β-sheets in which R1 labels of adjacent protein strands stack in parallel can be estimated by ESR line shape analysis. This is because, when packing in parallel β-sheet structures, the same R1-labeled sites from different protein strands come into intimate contact within 5–8 Å, yielding a characteristic single-line ESR spectral feature due to spin exchange between overlapping electron spin orbitals of the R1 labels (29, 36–41). We can verify the assignment of this spectral signature to parallel β-sheet stacking by systematic “spin dilution” to yield a mixture where only 20% of the tau strands are labeled with paramagnetic R1 labels and 80% are labeled with the diamagnetic analog of R1 labels, designated here as R1’, at a given site of Δtau187. This approach allows us to differentiate between the spin-exchanged and simply immobile spectral features, thereby confirming the origin of the characteristic spectral component as caused by β-sheet packing (36–41). Single-line, spin-exchanged, ESR spectra of protein fibrils have been previously assigned to parallel β-sheet structures (29, 36–41), including the mature fibrils of full-length tau proteins that were found to stack in parallel and in-register particularly around the PHF6 region (29).By combining ODNP and cw ESR measurements performed in situ and in solution together with conventional techniques of turbidimetry, Thioflavin T (ThT) staining fluorescence, transmission electron microscopy (TEM), and chemical cross-linking to probe aggregation of Δtau187, we aim to answer the following key questions: (i) At what stage of aggregation do structural transformations of tau proteins and their assembly occur? (ii) What are the structural and dynamic properties of the aggregation intermediates, and are they “on pathway” toward fibril formation? (iii) At what stage do stable β-sheet structures form, and what are their populations? (iv) Does fibril growth proceed mainly by elongation of preassembled oligomers or fibrils, or predominantly by monomer recruitment?