Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Definition of a third VLR gene in hagfish

Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish “VLRA” is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.Phylogenetic studies of immunity indicate the emergence of two types of recombinatorial adaptive immune systems (AISs) in vertebrates (1, 2). All of the extant jawed vertebrates generate a vast repertoire of Ig-domain–based T- and B-cell antigen receptors primarily by the recombinatorial assembly of Ig V-(D)-J gene segments and somatic hypermutation (3). The extant jawless vertebrates, lampreys and hagfish, instead have an alternative AIS that is based on variable lymphocyte receptors (VLRs), the diversity of which is generated through recombinatorial use of leucine-rich repeat (LRR) cassettes (4–6). The germ-line VLR genes are incomplete in that they only contain coding sequences for the leader sequence, incomplete amino- and carboxyl-terminal LRR subunits (LRRNT and LRRCT) and the stalk region (4, 7, 8). However, each germ-line VLR gene is flanked by hundreds of different LRR-encoding sequences, which can be used as templates to add LRR sequences during the assembly of a mature VLR gene (4, 8–11). This gene conversion-like process is postulated to involve the activation-induced cytidine deaminase (AID) orthologs, cytidine deaminases 1 and 2 (CDA1 and CDA2) (8, 12). The combinatorial VLR assembly can generate a vast repertoire of anticipatory receptors comparable in diversity to the repertoire of Ig-domain–based antigen receptors in jawed vertebrates (8, 9).Three VLR genes have been identified in lampreys (VLRA, VLRB, and VLRC), but only two (VLRA and VLRB) have been identified so far in hagfish (4, 7, 8, 13). Lamprey VLRB-expressing cells can respond to immunization by undergoing lymphoblastoid transformation, clonal expansion, and secretion of their antigen-specific VLRB antibodies (9, 14). VLRA- and VLRC-bearing cells also proliferate in response to antigen stimulation, but do not differentiate into antibody secreting cells; instead they maintain cell surface expression of their receptors, while increasing the expression of proinflammatory cytokines, macrophage migration inhibitory factor (MIF), and interleukin-17 (IL-17) (12). CDA1-expressing progenitors assemble their VLRA and VLRC genes to become VLRA⁺ and VLRC⁺ lymphocytes in a thymus-equivalent region of the gills termed the thymoid (15, 16). Conversely, VLRB assembly coincides with CDA2 expression during VLRB⁺ lymphocyte development in hematopoietic tissues (15). The T- and B-like characteristics of the lamprey lymphocytes imply that jawless vertebrates have humoral and cellular arms of adaptive immunity comparable to those of jawed vertebrates.In the present study, we sought to determine whether or not hagfish have a third VLR gene. In comparing the sequences of the two known hagfish VLRs with those of the three lamprey VLRs, we noticed that the highly variable inserts in the LRRCT module of the currently designated hagfish “VLRA” are more similar to those of lamprey VLRC than to those of lamprey VLRA (13, 17, 18). This led us to hypothesize that a third hagfish VLR, if present, would be the true counterpart of lamprey VLRA. Through a similarity search against the hagfish database, we identified a fragment of a potential third VLR gene, which was in turn used to clone and sequence a previously uncharacterized VLR gene. Here, we report the characterization of this third VLR type in pacific hagfish (Eptatretus stoutii) and its phylogenetic and structural relationship with previously identified VLRs. Our findings suggest a modified nomenclature for the hagfish VLRs.     

Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.The auxin binding protein 1 (ABP1) was first isolated from maize plants based on its ability to bind auxin (1). The crystal structure of ABP1 demonstrated clearly that ABP1 has an auxin-binding pocket and, indeed, binds auxin (2). However, the elucidation of the physiological functions of ABP1 has been challenging because the first reported abp1 T-DNA insertion mutant in Arabidopsis was not viable (3). Nevertheless, ABP1 has been recognized as an essential gene for plant development and as a key component in auxin signaling (4–9). Because viable abp1 null mutants in Arabidopsis were previously unavailable, alternative approaches have been used to disrupt ABP1 function in Arabidopsis to determine the physiological roles of the protein. Cellular immunization approaches were used to generate ABP1 knockdown plants (10, 11). Inducible overexpression of the single chain fragment variable regions (scFv12) of the anti-ABP1 monoclonal antibody mAb12 both in cell lines and in Arabidopsis plants presumably neutralizes the endogenous ABP1 activities (10, 11). Two such antibody lines, SS12S and SS12K, have been widely used in many ABP1-related studies (4, 6, 9–11). The results obtained from the characterization of the antibody lines suggest that ABP1 regulates cell division, cell expansion, meristem activities, and root development (4, 6, 10, 12, 13). Transgenic plants that overexpress ABP1 antisense RNA were also used to elucidate the physiological functions of ABP1 (4, 10). Moreover, missense point mutation alleles of abp1 have also been generated through the Arabidopsis TILLING project. One such TILLING mutant, named abp1-5, harbors a mutation (His94 >Tyr) in the auxin-binding pocket and has been widely used in many ABP1-related studies (4, 8, 9). Previous studies based on the antisense lines, antibody lines, and Arabidopsis mutant alleles have led to the conclusion that ABP1 is essential for embryogenesis, root development, and many other developmental processes. However, the interpretation of results generated by using the ABP1 antisense and antibody lines are not straightforward and off-target effects have not been completely ruled out. We believe that characterization of abp1 null plants is urgently needed to unambiguously define the roles of ABP1 in auxin signaling and in plant development.In the past several years, studies of the presumed ABP1-mediated auxin signal transduction pathway were carried out in several laboratories. It has been hypothesized that ABP1 is an auxin receptor mediating fast, nongenomic effects of auxin (4–6, 8, 9), whereas the TIR1 family of F-box protein/auxin receptors are responsible for auxin-mediated gene regulation (14, 15). One of the proposed functions of ABP1 is to regulate subcellular distribution of PIN auxin efflux carriers (6, 9, 13). Furthermore, a recent report suggests that a cell surface complex consisting of ABP1 and transmembrane receptor-like kinases functions as an auxin receptor at the plasma membrane by activating the Rho-like guanosine triphosphatases (GTPases) (ROPs) in an auxin-dependent manner (8). ROPs have been reported to play a role in regulating cytoskeleton organization and PIN protein endocytosis (5, 6). However, it is important to unequivocally determine the biological processes that require ABP1 before extensive efforts are directed toward elucidating any ABP1-mediated signaling pathways.In this paper, we generate and characterize new abp1 null mutants in Arabidopsis. We are interested in elucidating the molecular mechanisms by which auxin regulates flower development because our previously identified auxin biosynthetic mutants display dramatic floral defects (16–18). Because ABP1 was reported as an essential gene and ABP1 binds auxin (2, 3), we decided to determine whether ABP1 plays a role in flower development. We used our recently developed ribozyme-based CRISPR gene editing technology (19) to specifically inactivate ABP1 during flower development. Unexpectedly, we recovered a viable abp1 mutant (abp1-c1, c stands for alleles generated by using CRISPR) that contains a 5-bp deletion in the first exon of ABP1. We also isolated a T-DNA abp1 allele (abp1-TD1) that harbors a T-DNA insertion in the first exon of ABP1. We show that both abp1-c1 and abp1-TD1 are null mutants. Surprisingly, the mutants were indistinguishable from wild-type (WT) plants at all of the developmental stages we analyzed. Our data clearly demonstrate that ABP1 is not an essential gene and that ABP1 does not play a major role in auxin signaling and Arabidopsis development under normal growth conditions.     

Activation of Big Grain1 significantly improves grain size by regulating auxin transport in rice

Grain size is one of the key factors determining grain yield. However, it remains largely unknown how grain size is regulated by developmental signals. Here, we report the identification and characterization of a dominant mutant big grain1 (Bg1-D) that shows an extra-large grain phenotype from our rice T-DNA insertion population. Overexpression of BG1 leads to significantly increased grain size, and the severe lines exhibit obviously perturbed gravitropism. In addition, the mutant has increased sensitivities to both auxin and N-1-naphthylphthalamic acid, an auxin transport inhibitor, whereas knockdown of BG1 results in decreased sensitivities and smaller grains. Moreover, BG1 is specifically induced by auxin treatment, preferentially expresses in the vascular tissue of culms and young panicles, and encodes a novel membrane-localized protein, strongly suggesting its role in regulating auxin transport. Consistent with this finding, the mutant has increased auxin basipetal transport and altered auxin distribution, whereas the knockdown plants have decreased auxin transport. Manipulation of BG1 in both rice and Arabidopsis can enhance plant biomass, seed weight, and yield. Taking these data together, we identify a novel positive regulator of auxin response and transport in a crop plant and demonstrate its role in regulating grain size, thus illuminating a new strategy to improve plant productivity.Because it is one of the most important staple food crops cultivated worldwide, improvement of grain yield is a major focus of rice-breeding programs (1). Grain size is one of the determining factors of grain yield (2, 3). A number of quantitative trait loci (QTLs) controlling rice grain size have been identified in recent years (4–11). However, functional mechanisms of these genes remain largely unknown. Because QTLs usually have important functions in determining grain size, many of them have been widely selected in breeding processes or existed in modern elite varieties, and a certain QTL could be only applicable in certain varieties (12). Thus, exploration of new grain size-associated genes and elucidation of their functional mechanisms have great significance for further improvement of rice yield (12).Seed size, as well as other organ size, is controlled by various plant hormones, such as auxin, brassinosteroid, and cytokinin (10, 13, 14). A number of studies have demonstrated that auxin plays a vital role in organ size determination by affecting cell division, cell expansion, and differentiation (15–17). Auxin exists predominantly as indole-3-acetic acid (IAA) in plants, and genetic studies of its biosynthetic genes in Arabidopsis have demonstrated that IAA regulates many aspects of plant growth and development, including stem elongation, lateral branching, vascular development, and tropic growth responses (18, 19). Combined with biochemical studies, the tryptophan (Trp)-dependent IAA biosynthesis pathway has been clearly established involving the YUCCA family flavin monooxgenases (20). Importantly, the two-step pathway is highly conserved throughout the plant kingdom (21). Until very recently, the Trp-independent auxin biosynthetic pathway was elucidated as contributing to early embryogenesis in Arabidopsis (22). Primary auxin signaling is a rapid process initiated from the hormone perception by receptor TIR1, an F-box protein, followed by degradation of the negative regulator AUX/IAA proteins, and further release the downstream auxin response factors (ARFs) (23–26). However, how the ARFs work in plants remains elusive. Auxin transport, generally referring to the cell-to-cell transportation of the hormone directed basipetally from shoots to roots in vascular tissues, plays a critical role in auxin response (18). The transport involves a number of membrane-associated proteins, such as PINs (protein inhibitor of nNOS), AUX1 (AUXIN TRANSPORTER PROTEIN 1), and ABCBs (ATP-BINDING CASSETTE, SUB-FAMILY B PROTEINS) as efflux or influx carriers (27–30). Disruption of auxin transport induced by either gene mutations or chemical inhibitor treatment will lead to diverse development defects, such as decreased lateral organ initiation and defective tropic growth responses (27, 31–34).In this study, we identify a rice mutant, named big grain1-D (Bg1-D) because it is a dominant mutant having extralarge grain size. BG1 encodes a novel plasma membrane-associated protein, and is specifically induced by auxin treatment. We show that BG1 is a new positive regulator of auxin response involved in auxin transport, and demonstrate that manipulation of BG1 expression can greatly improve grain size and plant productivity.     

Dynamics of gene circuits shapes evolvability

To what extent does the dynamical mechanism producing a specific biological phenotype bias the ability to evolve into novel phenotypes? We use the interpretation of a morphogen gradient into a single stripe of gene expression as a model phenotype. Although there are thousands of three-gene circuit topologies that can robustly develop a stripe of gene expression, the vast majority of these circuits use one of just six fundamentally different dynamical mechanisms. Here we explore the potential for gene circuits that use each of these six mechanisms to evolve novel phenotypes such as multiple stripes, inverted stripes, and gradients of gene expression. Through a comprehensive and systematic analysis, we find that circuits that use alternative mechanisms differ in the likelihood of reaching novel phenotypes through mutation. We characterize the phenotypic transitions and identify key ingredients of the evolutionary potential, such as sensitive interactions and phenotypic hubs. Finally, we provide an intuitive understanding on how the modular design of a particular mechanism favors the access to novel phenotypes. Our work illustrates how the dynamical mechanism by which an organism develops constrains how it can evolve. It is striking that these dynamical mechanisms and their impact on evolvability can be observed even for such an apparently simple patterning task, performed by just three-node circuits.Evolution occurs through mutations on existing genotypes, potentially transforming the original phenotype or trait into a novel one, with latent beneficial consequences. It is a fundamental problem in biology to understand the relationship between a genotype and the associated phenotypes accessible through mutations, in other words, its evolvability. From the many definitions of evolvability (1, 2), we refer here to the ability of genotypes to access novel phenotypes, irrespective of the subsequent process of natural selection.To understand how a phenotype evolves we need to consider that a huge number of distinct genotypes can achieve that same phenotype. For example, hundreds of distinct RNA sequences fold in the same secondary structure (3), as do proteins in their 3D structure (4). Similarly, distinct gene regulatory architectures can produce the same gene expression pattern (5, 6) or temporal behavior (7, 8). However, among these genotypes, some are more evolvable than others. The existing studies have targeted two key drivers of evolvability: a genotype’s design and a genotype’s location within a neutral space.A first class of studies focuses on a circuit’s general architectural features, such as feed-back or feed-forward loops, revealing that these distinct families of designs or motifs differ in their evolvability (9, 10). The second class of studies centers not on single designs but on the whole collection of genotypes capable of producing the same phenotype. These genotypes with a common phenotype form a region in genotype space termed a neutral space or neutral network (3), as mutations within this region produce no change in the phenotype.As revealed by many studies, the existence of neutral spaces has two major consequences to the evolutionary process. First, these neutral spaces often appear as fully connected and dense regions (11–13). Therefore, although genotypes internal to the neutral space are highly robust to mutations (i.e., not evolvable), only genotypes close to the edges of the neutral space might access novel phenotypes. From this perspective, neutral mutations and thus the process of neutral drift can generate cryptic genetic variation (14) by moving a species closer to the edges of the neutral space into a more evolvable state (12, 15). Second, different positions in genotype space give access to distinct novel phenotypes. Large neutral spaces percolate through genotype space, providing access to a diversity of novel phenotypes from different genotypes (11–13). In a nutshell, the accessible innovations are critically determined by a genotype’s position in genotype space (16) (Fig. 1).Open in a separate windowFig. 1.Phenotype-based view on evolvability. (A) Evolvability accounts for the accessible novel phenotypes, whereas developmental constraints imply that certain hypothetical forms are not possible: phenotype 2 (purple) is not available by gradual mutation. (B) Innovations accessible from a given genotype constitute its phenotypic neighborhood. The arrangement and diversity of this neighborhood is a measure of the genotype’s evolvability (16). Genotype space is high dimensional, but we schematically represent it here in 2D for illustrative purposes.Although the abovementioned features of genotype-phenotype maps have been much studied, another important aspect of the system has thus far been neglected. None of the existing studies addressed the impact of the underlying dynamical mechanism of a gene circuit on its evolvability. By mechanism, we mean the causal dynamics responsible for the trajectory of the system (i.e., the spatiotemporal course of gene expression) resulting in the final phenotype. In addition to the increasing awareness that dynamics itself is a decisive property of gene circuits (17), several specific observations led us to hypothesize that dynamics does impact on evolvability.First, to achieve a given biological function, a gene circuit uses one of few fundamental solutions referred to as dynamical mechanisms (5–7, 18–20). More specifically, circuits with the same underlying dynamical mechanism share a common arrangement of phase portraits (20, 21). Second, Cotterell and Sharpe (6) revealed that, for a simple patterning function, it is not possible to smoothly and functionally transition from one mechanism to another. That is, in contrast to the common view (11–13), this particular neutral space does not form a single fully connected region when the underlying mechanism is taken into account. Instead, the neutral space for the simple patterning function studied by Cotterell and Sharpe (6) breaks up into scattered islands of genotypes characterized by distinct underlying mechanisms. These observations suggest that evolvability may be constrained specifically by the dynamical mechanism of the gene circuit. As neutral spaces can be broken up into a discrete collection of separated islands, the process of neutral drift may be limited to these mechanism-specific regions.To assess the impact of dynamical mechanisms, we chose to study circuits that control spatial (multicellular) gene expression patterns. It is well established in developmental biology that the spatial organization of gene expression orchestrates cell differentiation. Their diversification causes evolution of both modest morphological traits, such as novel pigmentation patterns (22), and major evolutionary breakthroughs, such as new body structures (23). Here we chose to address the interpretation of a morphogen gradient by a field of cells into different cell fates (5–7, 18, 24–27) (Fig. 2A), a critical patterning event in embryo’s morphogenesis (28). We build on the work of Cotterell and Sharpe (6), who extracted six fundamental mechanisms for this patterning task: Bistable, Incoherent feed-forward, Mutual Inhibition, Overlapping Domains, Classical, and Frozen Oscillator (Fig. 2B and SI Appendix, Fig. S1).Open in a separate windowFig. 2.Alternative mechanisms to achieve a single phenotype. (A) Within the French Flag conceptual framework, a preestablished fixed concentration gradient (input) is interpreted by a one-dimensional row of cells into different cell fates through a threshold-dependent mechanism. Additionally, cells communicate to one another through diffusive gene products (dashed arrows). We exhaustively enumerate all possible three-gene circuit topologies and sample large numbers of genotypes (i.e., parameter values; SI Appendix, Methods). Solutions of our search are genotypes able to interpret the morphogen gradient into a band of gene expression (6). Similar exhaustive approaches have being adopted for exploring a variety of biological functions, such as temporal behaviors (7, 25) or other spatial patterning functions (5, 18). (B) A stripe-forming circuit uses one of six distinct mechanisms (6), each mechanism uses a distinct gene expression dynamics in space and time to reach the same phenotype. Importantly, Mutual Inhibition (bicoid-hunchback-knirps), Incoherent feed-forward (caudal-knirps-giant), and Classical (hunchback-krüppel-knirps) are involved in Drosophila anterior-posterior patterning (26), whereas Incoherent feed-forward controls the mesoderm inducer Xenopus Brachyury (27).For the current study, we analyzed each of these six mechanisms independently and obtained a mechanism-specific measure of evolvability. We found that, indeed, the likelihood of accessing distinct phenotypic innovations is different for each dynamical mechanism, despite the fact that they all produce the same phenotype. Our analysis uncovers key features of the mechanistic neutral spaces and provides useful insight into how phenotypic transitions and thus innovations occur.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

Spatiotemporal dynamics of gene flow and hybrid fitness between the M and S forms of the malaria mosquito,Anopheles gambiae

The M and S forms of Anopheles gambiae have been the focus of intense study by malaria researchers and evolutionary biologists interested in ecological speciation. Divergence occurs at three discrete islands in genomes that are otherwise nearly identical. An “islands of speciation” model proposes that diverged regions contain genes that are maintained by selection in the face of gene flow. An alternative “incidental island” model maintains that gene flow between M and S is effectively zero and that divergence islands are unrelated to speciation. A “divergence island SNP” assay was used to explore the spatial and temporal distributions of hybrid genotypes. Results revealed that hybrid individuals occur at frequencies ranging between 5% and 97% in every population examined. A temporal analysis revealed that assortative mating is unstable and periodically breaks down, resulting in extensive hybridization. Results suggest that hybrids suffer a fitness disadvantage, but at least some hybrid genotypes are viable. Stable introgression of the 2L speciation island occurred at one site following a hybridization event.The M and S forms of the African malaria vector Anopheles gambiae have been the subject of intense study over the past decade. The focus has centered on models of the evolution and maintenance of genetic divergence between the two forms in relation to speciation (reviewed in ref. 1). A. gambiae has become a model, described in a number of recent reviews on speciation (2–5).The two forms occur in sympatry throughout West and Central Africa (6). They were initially described on the basis of several single nucleotide polymorphisms (SNPs) in the X-linked ribosomal DNA locus (7, 8). Heterozygotes were rarely found in nature and studies of reproductive isolation (RI) confirmed strong assortative mating with interform matings occurring at a frequency of ∼1% (9). Progeny of laboratory crosses and backcrosses show no signs of reduced fitness (10). However, it is widely held that, in nature, some degree of ecologically dependent postzygotic isolation, in addition to assortative mating, contributes to divergence between the two forms (11, 12).Studies of the genetic structure of M and S based on microsatellite markers revealed little between-form differentiation outside the centromeric region of the X chromosome and a few regions associated with inversions (13–15). This overall lack of divergence was attributed to the homogenizing effect of gene flow between the forms (16).In 2008, several reports of much higher frequencies of M/S hybrids, as high as 24%, appeared (17–19). These were all observed in populations in coastal West Africa, an area now thought to represent a zone of secondary contact (20, 21). These reports resulted in the emergence of this species as the focus of research aimed at exploring the evolution and maintenance of genetic divergence with gene flow (11, 12, 22, 23).The first genome-wide comparison of the M and S forms by Turner et al. (24) was consistent with earlier observations that divergence overall is low, but there are small, discrete regions of divergence representing about 3% of the genome. They identified three diverged regions: one near the centromere on the X chromosome, one on the left arm of chromosome 2 (2L), and one on the right arm of chromosome 2 (2R). A number of genome-wide scans comparing M and S have been conducted since. These have applied several methods, including the same microarray used by Turner et al. (25, 26), high-density SNP arrays (12, 21, 23, 27), and whole-genome sequencing (28). These studies likewise revealed little divergence except in a few discrete regions of the genome characterized by high levels of differentiation (islands of divergence).This body of work has culminated in two opposing models aimed at describing the evolution of M and S (1, 11). The “islands of speciation” model supposes that (i) small regions of divergence contain genes responsible for RI because they are directly associated with assortative mating and/or are under strong ecologically dependent divergent selection, and (ii) the rest of the genome is either neutral with respect to differentiation or close enough to neutral so that contemporary gene flow overwhelms selection. The alternative “incidental island” model recognizes the presence of islands of divergence, but suggests that (i) these are not related to RI and the remainder of the genome is less differentiated due to segregating ancestral polymorphism, not gene flow, and (ii) F₁ hybrids are effectively sterile, therefore the amount of “realized” gene flow between M and S is near zero and that M and S are in fact “good species.” Indeed, the M form has recently been elevated to species status and provided the formal species name Anopheles coluzzii (29). We continue to refer to M and S forms to facilitate discussion with reference to the recent literature.Limitations in the genome-wide scans described above may have contributed to disparate views of the evolution of divergence between A. gambiae subgroups. All comparisons of natural populations to date used single-locus, X-linked genotypes (7, 30–32) to identify M and S form specimens used in downstream analyses. These single-locus assays misidentify a significant proportion of backcross individuals. In addition, assessment of hybridization frequencies and comprehensive tests for introgression are precluded from these studies because DNA pools were used (21, 23, 27), sample sizes were too small (24, 26), or such assessments may be irrelevant because M and S laboratory colony mosquitoes were used (28). Finally, there are strong limitations in relying on genome scans alone for detecting selection, gene flow, and recombination that occurs during speciation (33, 34).In this study, we used a simple multilocus SNP genotype approach to distinguish M, S, F₁ hybrids, and backcross individuals (35), which we call the “divergence island SNP” (DIS) assay (SI Appendix). We describe the spatial and temporal distribution of hybridization frequencies, the extent to which hybridization results in introgression, and the fitness of hybrid genotypes in nature. Assumptions concerning these between-form interactions have played a central role in the interpretation of comparative genomics data as applied to understanding speciation in this system. The results we report challenge a number of these assumptions and they provide unique information about temporal dynamics in the way in which M and S forms interact that suggests new avenues for future research.     

Conditional ablation of HMGB1 in mice reveals its protective function against endotoxemia and bacterial infection

High-mobility group box 1 (HMGB1) is a DNA-binding protein abundantly expressed in the nucleus that has gained much attention for its regulation of immunity and inflammation. Despite this, whether and how HMGB1 contributes to protective and/or pathological responses in vivo is unclear. In this study, we constructed Hmgb1-floxed (Hmgb1^f/f) mice to achieve the conditional inactivation of the gene in a cell- and tissue-specific manner by crossing these mice with an appropriate Cre recombinase transgenic strain. Interestingly, although mice with HMGB1 ablation in myeloid cells apparently develop normally, they are more sensitive to endotoxin shock compared with control mice, which is accompanied by massive macrophage cell death. Furthermore, these mice also show an increased sensitivity to Listeria monocytogenes infection. We also provide evidence that the loss of HMGB1 in macrophages results in the suppression of autophagy, which is commonly induced by lipopolysaccharide stimulation or L. monocytogenes infection. Thus, intracellular HMGB1 contributes to the protection of mice from endotoxemia and bacterial infection by mediating autophagy in macrophages. These newly generated HMGB1 conditional knockout mice will serve a useful tool with which to study further the in vivo role of this protein in various pathological conditions.Of the four members of the high-mobility group box (HMGB) family, HMGB1 is the best studied, given its versatile functions in various aspects of cellular responses (1–5). Ubiquitously expressed in all cells, HMGB1 is found en masse in the nucleus and is supposedly released into the extracellular fluid through an endoplasmic reticulum–Golgi pathway-independent mechanism from immune cells such as monocytes or macrophages after stimulation with lipopolysaccharide (LPS), proinflammatory cytokines, or nitric oxide (1, 6). The release of HMGB1 is also regulated by the inflammasome, a multiprotein oligomer that activates caspase-1 to promote the maturation of inflammatory cytokines, interleukin-1β (IL-1β) and IL-18, and by dying cells, typically those undergoing necrosis (7–10). Secreted or released, HMGB1 is known to participate in the activation of cell surface innate immune receptors, typically Toll-like receptors (TLRs), thereby affecting many aspects of the host’s inflammatory responses upon infection or noxious stresses (1–5). Perhaps most notably is the crucial role of HMGB1 in LPS-induced endotoxemia, whereby administration of an anti-HMGB1 antibody significantly protects mice from lethality (1, 11). The study of released HMGB1 is complicated by a number of complex posttranslational modifications made to the protein, including acetylation and redox modifications that may regulate HMGB1 function (12–14).HMGB1 can regulate immune reactions in several ways. Cytosolic HMGB1, together with the other members of the family, function as universal sentinels or chaperones for immunogenic nucleic acids by facilitating the recognition of nucleic acids by more discriminative, nucleic acid-sensing innate receptors (15–17). In addition, HMGB1 regulates autophagy, a cellular response that functions in clearing long-lived proteins and dysfunctional organelles to generate substrates for adenosine triphosphate (ATP) production during periods of starvation and other types of cellular stress events (13, 18–20). This mechanism contributes to antimicrobial responses against invading microorganisms (21, 22). Indeed, microorganisms can induce autophagy by stimulating innate immune receptors, such as TLRs, by a process in which bacteria are captured by phagocytosis but remain within intact vacuoles, an autophagic process termed microtubule-associated protein light chain 3 (LC3)-associated phagocytosis (LAP), which promotes the maturation of autophagosomes into autolysosomes (23, 24).Collectively, these studies place HMGB1 in the center of immunological events where it uniquely functions intracellularly and extracellularly as a mediator of immune and inflammatory responses. The biological and clinical importance of HMGB1 is underscored by the dysregulation of this protein in a number of pathological conditions, including sepsis, ischemia–reperfusion injury, arthritis, and cancer (1, 3–5). Nonetheless, in vivo validation of the versatile functions described above is lacking due to the lethality of the Hmgb1-deficient mice, thought to cause lethal hypoglycemia in newborn mice (25). In the present study, we describe the generation of Hmgb1-floxed (Hmgb1^f/f) mice that enabled the cell- and tissue-specific deletion of the gene when crossed with an appropriate Cre recombinase transgenic strain. We demonstrate in this study a protective role of intracellular HMGB1 in macrophages where it serves as a crucial regulator of autophagosome formation in the context of LPS stimulation or bacterial infection in vivo. Finally, we will discuss the future prospects of HMGB1 research using these newly generated mutant mice.     

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field

Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana. To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics of A. thaliana.The genomes of eukaryotes originate from ancient endosymbiotic associations that eventually led to energy-harnessing organelles: mitochondria, common to all eukaryotes, and chloroplasts in the “green” lineage. The evolution of endosymbionts into cellular organelles was accompanied by massive gene loss, with a large proportion being transferred to the nucleus (1, 2). Nevertheless, mitochondria and chloroplasts retained a few (30–80) protein-encoding genes that play crucial roles in energy metabolism (respiration and photosynthesis). Mitochondrion and chloroplast metabolisms rely on the proper interaction of nuclear-encoded proteins and their counterparts encoded in the organelle genome. Consequently, the genes in nuclear and organellar compartments are expected to be coadapted (3).Cytonuclear coadaptation has been demonstrated by altered phenotypes observed on interspecific exchanges of cytoplasm between related species in mammals (4), yeast (5), arthropods (6), and plants, whose interspecific crosses are frequently successful (7). These alterations affect organelle function and even the organism phenotype, indicating epistasis between nuclear and cytoplasmic genes. Although cytonuclear coadaptation is generally studied at the interspecific level, the existence of intraspecific genetic diversity in organelle genomes suggests a potential for genomic coadaptation within species. A few studies have reported phenotypic effects of intraspecific cytonuclear epistasis in nonplant species (8–11). In plants, many studies have focused on cytoplasmic male sterility (CMS), an impairment of pollen production governed by nucleo-mitochondrial interactions in some hermaphroditic species (12), in particular in crops and their relatives (13). The phenotypic effects of intraspecific cytonuclear epistasis other than CMS have been reported in only a limited number of plant systems (14–17), with evidence that cytoplasmic variation contributes to local adaptation (18, 19).In recent years, several studies using reciprocal segregating populations of the model plant Arabidopsis thaliana have investigated the effect of cytonuclear epistasis on a number of laboratory-measured phenotypes such as the metabolome, defense chemistry and growth (17, 20, 21), water-use efficiency (22, 23), and seed germination (24, 25). Although some studies have reported significant effects of cytonuclear epistasis (17, 20, 21, 23, 25), others have found additive cytoplasmic effects but with weak or no cytonuclear epistasis (22). Each of these studies (with the exception of ref. 25) was, however, based on a single reciprocal cross between two natural accessions, thereby preventing the estimation of the prevalence of cytonuclear epistasis in this species. In addition, although these reports involve adaptive traits (26–30), the investigation of the effect of cytonuclear epistasis on adaptive phenotypes in field conditions is, at best, scarce in A. thaliana.Here, following the modern standards of ecological genomics (31), we explored the prevalence of cytonuclear interactions on adaptive whole-organism traits in the model plant A. thaliana in a field experiment. To do so, based on eight natural accessions of a core collection that covers a significant part of the species’ cytoplasmic and nuclear genetic diversity in A. thaliana (25, 32), we created eight series of seven cytolines. Cytolines are genotypes that combine the nuclear genome from one parent with the organelle genomes of another (33). We examined the cytolines and their parental accessions for effects of cytonuclear interactions on 28 field-measured traits related to germination, phenology, resource acquisition, plant architecture and seed dispersal, fecundity, and survival.     

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.The bacterium Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSIs), including cellulitis, furunculosis, and folliculitis (1–4), and a common etiologic agent of impetigo (5), erysipelas (6), and superinfection in atopic dermatitis (7). This bacterium is a significant cause of surgical or traumatic wound infections (8, 9), as well as decuibitus and diabetic skin lesions (10). Moreover, SSSI is an important risk factor for systemic infection. The skin is a key portal of entry for hematogenous dissemination, particularly in association with i.v. catheters. S. aureus is now the second most common bloodstream isolate in healthcare settings (11), and SSSI is a frequent source of invasive infections such as pneumonia or endocarditis (12, 13). Despite a recent modest decline in rates of methicillin-resistant S. aureus (MRSA) infection in some cohorts (13), infections due to S. aureus remain a significant problem (14, 15). Even with appropriate therapy, up to one-third of patients diagnosed with S. aureus bacteremia succumb—accounting for more attributable annual deaths than HIV, tuberculosis, and viral hepatitis combined (16).The empiric use of antibiotics in healthcare-associated and community-acquired settings has increased S. aureus exposure to these agents, accelerating selection of resistant strains. As a result, resistance to even the most recently developed agents is emerging at an alarming pace (17, 18). The impact of this trend is of special concern in light of high rates of mortality associated with invasive MRSA infection (e.g., 15–40% in bacteremia or endocarditis), even with the most recently developed antistaphylococcal therapeutics (19, 20). Moreover, patients who experience SSSI due to MRSA exhibit high 1-y recurrence rates, often prompting surgical debridement (21) and protracted antibiotic treatment.Infections due to MRSA are a special concern in immune-vulnerable populations, including hemodialysis (22), neutropenic (23, 24), transplantation (25), and otherwise immunosuppressed patients (26, 27), and in patients with inherited immune dysfunctions (28–31) or cystic fibrosis (32). Patients having deficient interleukin 17 (IL-17) or IL-22 responses (e.g., signal transduction mediators STAT3, DOCK8, or CARD9 deficiencies) exhibit chronic or “cold” abscesses, despite high densities of pathogens such as S. aureus (33, 34). For example, patients with Chronic Granulomatous Disease (CGD; deficient Th1 and oxidative burst response) have increased risk of disseminated S. aureus infection. In contrast, patients with Job’s Syndrome (deficient Th17 response) typically have increased risk to SSSI and lung infections, but less so for systemic S. aureus bacteremia (35, 36). This pattern contrasts that observed in neutropenic or CGD patients (37). These themes suggest efficacious host defenses against MRSA skin and invasive infections involve complementary but distinct molecular and cellular immune responses.From these perspectives, vaccines or immunotherapeutics that prevent or lessen severity of MRSA infections, or that enhance antibiotic efficacy, would be significant advances in patient care and public health. However, to date, there are no licensed prophylactic or therapeutic vaccine immunotherapies for S. aureus or MRSA infection. Unfortunately, efforts to develop vaccines targeting S. aureus capsular polysaccharide type 5 or 8 conjugates, or the iron-regulated surface determinant B protein, have not been successful thus far (38, 39). Likewise, passive immunization using monoclonal antibodies targeting the S. aureus adhesin clumping factor A (ClfA, tefibazumab) (40) or lipoteichoic acid (pagibaximab) (41) have not shown efficacy against invasive infections in human clinical studies to date. Moreover, the striking recurrence rates of SSSI due to MRSA imply that natural exposure does not induce optimal preventive immunity or durable anamnestic response to infection or reinfection. Thus, significant challenges exist in the development of an efficacious vaccine targeting diseases caused by S. aureus (42) that are perhaps not optimally addressed by conventional approaches.The NDV-3 vaccine reflects a new strategy to induce durable immunity targeting S. aureus. Its immunogen is engineered from the agglutinin-like sequence 3 (Als3) adhesin/invasin of Candida albicans, which we discovered to be a structural homolog of S. aureus adhesins (43). NDV-3 is believed to cross-protect against S. aureus and C. albicans due to sequence (T-cell) and conformational (B-cell) epitopes paralleled in both organisms (44). Our prior data have shown that NDV-3 is efficacious in murine models of hematogenous and mucosal candidiasis (45), as well as S. aureus bacteremia (46–48). Recently completed phase I clinical trials demonstrate the safety, tolerability, and immunogenicity of NDV-3 in humans (49).     

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.Faithful distribution of the duplicated genome into two daughter cells during mitosis depends on proper kinetochore–microtubule (MT) attachments. Defects in kinetochore–MT attachments result in chromosome missegregation, causing aneuploidy, a hallmark of cancer (1, 2). To ensure accurate chromosome segregation, cells use the spindle assembly checkpoint (SAC) to monitor kinetochore biorientation and to control the metaphase-to-anaphase transition. Cells enter anaphase only after the SAC is satisfied, requiring that all kinetochores be attached to MTs and be properly bioriented (3, 4). The core components of SAC signaling include mitotic arrest deficient-like 1 (Mad1), Mad2, Mad3/BubR1 (budding uninhibited by benzimidazole-related 1), Bub1, Bub3, monopolar spindle 1 (Mps1), and aurora B. The full SAC function requires the correct centromere/kinetochore localization of all SAC proteins (5).Among the SAC components, Mps1 was identified originally in budding yeast as a gene required for duplication of the spindle pole body (6). Subsequently, Mps1 orthologs were found in various species, from fungi to mammals. The stringent requirement of Mps1 for SAC activity is conserved in evolution (6–13). Human Mps1 kinase (also known as “TTK”) is expressed in a cell-cycle–dependent manner and has highest expression levels and activity during mitosis. Its localization is also dynamic (8, 14). Although the molecular mechanism remains unclear, Mps1 is required to recruit Mad1 and Mad2 to unattached kinetochores, supporting its essential role in SAC activity (15–18). It also is clear that aurora B kinase activity and the outer-layer kinetochore protein nuclear division cycle 80 (Ndc80)/Hec1 are required for Mps1 localization to kinetochores, as evidenced by recent work, including ours (17, 19–24). How Mps1 activates the SAC is now becoming clear. Mps1 recruits Bub1/Bub3 and BubR1/Bub3 to kinetochores through phosphorylation of KNL1, the kinetochore receptor protein of Bub1 and BubR1 (25–30).Despite much progress in understanding Mps1 functions, it remains unclear how Mps1 is involved in regulating chromosome alignment. In budding yeast mitosis, Mps1 regulates mitotic chromosome alignment by promoting kinetochore biorientation independently of Ipl1 (aurora B in humans) (31), but in budding yeast meiosis Mps1 must collaborate with Ipl1 to mediate meiotic kinetochore biorientation (32). In humans, Mps1 regulates chromosomal alignment by modulating aurora B kinase activity (33), but recent chemical biology studies show that Mps1 kinase activity is important for proper chromosome alignment and segregation, independently of aurora B (22, 34–36). Therefore whether Mps1 regulates chromosome alignment through modulation of aurora B kinase activity is still under debate (37).In this study, we reexamined the function of human Mps1 in chromosome alignment. We found that chromosomal alignment is largely achieved in Mps1 knockdown cells, provided that cells are arrested in metaphase in the presence of MG132, a proteasome inhibitor. However, disrupting Mps1 activity via small molecule inhibitors perturbs chromosomal alignment, even in the presence of MG132. This chromosome misalignment is caused by the abnormal accumulation of inactive Mps1 in the kinetochore and the subsequent failure of correct kinetochore–MT attachments. Further, we demonstrate that inactive Mps1 does not depend on the previously reported tetratricopeptide repeat (TPR) domain for localizing to kinetochores, and we identify a previously unidentified region adjacent to the C terminus of the TPR domain that is responsible for localizing inactive Mps1 to kinetochores. Thus, our work highlights that Mps1 kinase activity is necessary in regulating chromosome alignment and that it must be tightly regulated in space and time to ensure proper localization of Mps1 at kinetochores.