PFTAIRE Kinase L63 Interactor 1A (Pif1A Protein) Is Required for Actin Cone Movement during Spermatid Individualization in Drosophila melanogaster.

A useful model for determining the mechanisms by which actin and actin binding proteins control cellular architecture is the Drosophila melanogaster process of spermatogenesis. During the final step of spermatogenesis, 64 syncytial spermatids individualized as stable actin cones move synchronously down the axonemes and remodel the membranes. To identify new genes involved in spermatid individualization, we screened a collection of Drosophila male-sterile mutants and found that, in the line Z3-5009, actin cones formed near to the spermatid nuclei but failed to move, resulting in failed spermatid individualization. However, we show by phalloidin actin staining, electron microscopy and immunocytochemical localization of several actin binding proteins that the early cones had normal structure. We sequenced the genome of the Z3-5009 line and identified mutations in the PFTAIRE kinase L63 interactor 1A (Pif1A) gene. Quantitative real-time PCR showed that Pif1A transcript abundance was decreased in the mutant, and a transgene expressing Pif1A fused to green fluorescent protein (GFP) was able to fully rescue spermatid individualization and male fertility. Pif1A-GFP localized to the front of actin cones before initiation of movement. We propose that Pif1A plays a pivotal role in directing actin cone movement.

Adolescent Dopamine Neurons Represent Reward Differently during Action and State Guided Learning.

Neuronal underpinning of learning cause-and-effect associations in the adolescent brain remains poorly understood. Two fundamental forms of associative learning are Pavlovian (classical) conditioning, where a stimulus is followed by an outcome, and operant (instrumental) conditioning, where outcome is contingent on action execution. Both forms of learning, when associated with a rewarding outcome, rely on midbrain dopamine neurons in the ventral tegmental area (VTA) and substantia nigra (SN). We find that, in adolescent male rats, reward-guided associative learning is encoded differently by midbrain dopamine neurons in each conditioning paradigm. Whereas simultaneously recorded VTA and SN adult neurons have a similar phasic response to reward delivery during both forms of conditioning, adolescent neurons display a muted reward response during operant but a profoundly larger reward response during Pavlovian conditioning. These results suggest that adolescent neurons assign a different value to reward when it is not gated by action. The learning rate of adolescents and adults during both forms of conditioning was similar, supporting the notion that differences in reward response in each paradigm may be because of differences in motivation and independent of state versus action value learning. Static characteristics of dopamine neurons, such as dopamine cell number and size, were similar in the VTA and SN of both ages, but there were age-related differences in stimulated dopamine release and correlated spike activity, suggesting that differences in reward responsiveness by adolescent dopamine neurons are not because of differences in intrinsic properties of these neurons but engagement of different dopaminergic networks.SIGNIFICANCE STATEMENT Reckless behavior and impulsive decision-making by adolescents suggest that motivated behavioral states are encoded differently by the adolescent brain. Motivated behavior, which is dependent on the function of the dopamine system, follows learning of cause-and-effect associations in the environment. We find that dopamine neurons in adolescents encode reward differently depending on the cause-and-effect relationship of the means to receive that reward. Compared with adults, reward contingent on action led to a muted response, whereas reward that followed a cue but was not gated by action produced an augmented phasic response. These data demonstrate an age-related difference in dopamine neuron response to reward that is not uniform and is guided by processes that differentiate between state and action values.

Sex and strain differences in dynamic and static properties of the mesolimbic dopamine system.

Sex is a biological variable that contributes to the incidence, clinical course, and treatment outcome of brain disorders. Chief among these are disorders associated with the dopamine system. These include Parkinson's disease, ADHD, schizophrenia, and mood disorders, which show stark differences in prevalence and outcome between men and women. In order to reveal the influence of biological sex as a risk factor in these disorders, there is a critical need to collect fundamental information about basic properties of the dopamine system in males and females. In Long Evans rats, we measured dynamic and static properties related to the mesolimbic dopamine system. Static measures included assessing ventral tegmental area (VTA) dopamine cell number and volume and expression of tyrosine hydroxylase and dopamine transporter. Dynamic measures in behaving animals included assessing (1) VTA neuronal encoding during learning of a cue-action-reward instrumental task and (2) dopamine release in the nucleus accumbens in response to electrical stimulation of the VTA, vesicular depletion of dopamine, and amphetamine. We found little or no sex difference in these measures, suggesting sexual congruency in fundamental static and dynamic properties of dopamine neurons. Thus, dopamine related sex-differences are likely mediated by secondary mechanisms that flexibly influence the function of the dopamine cells and circuits. Finally, we noted that most behavioral sex differences had been reported in Sprague-Dawley rats and repeated some of the above measures in that strain. We found some sex differences in those animals highlighting the importance of considering strain differences in experimental design and result interpretation.

Sex differences in reward- and punishment-guided actions.

Differences in the prevalence and presentation of psychiatric illnesses in men and women suggest that neurobiological sex differences confer vulnerability or resilience in these disorders. Rodent behavioral models are critical for understanding the mechanisms of these differences. Reward processing and punishment avoidance are fundamental dimensions of the symptoms of psychiatric disorders. Here we explored sex differences along these dimensions using multiple and distinct behavioral paradigms. We found no sex difference in reward-guided associative learning but a faster punishment-avoidance learning in females. After learning, females were more sensitive than males to probabilistic punishment but less sensitive when punishment could be avoided with certainty. No sex differences were found in reward-guided cognitive flexibility. Thus, sex differences in goal-directed behaviors emerged selectively when there was an aversive context. These differences were critically sensitive to whether the punishment was certain or unpredictable. Our findings with these new paradigms provide conceptual and practical tools for investigating brain mechanisms that account for sex differences in susceptibility to anxiety and impulsivity. They may also provide insight for understanding the evolution of sex-specific optimal behavioral strategies in dynamic environments.

Androgen Regulation of the Mesocorticolimbic System and Executive Function.

Multiple lines of evidence indicate that androgens, such as testosterone, modulate the mesocorticolimbic system and executive function. This review integrates neuroanatomical, molecular biological, neurochemical, and behavioral studies to highlight how endogenous and exogenous androgens alter behaviors, such as behavioral flexibility, decision making, and risk taking. First, we briefly review the neuroanatomy of the mesocorticolimbic system, which mediates executive function, with a focus on the ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC). Second, we present evidence that androgen receptors (AR) and other steroid receptors are expressed in the mesocorticolimbic system. Using sensitive immunohistochemistry and quantitative polymerase chain reaction (qPCR) techniques, ARs are detected in the VTA, NAc, mPFC, and OFC. Third, we describe recent evidence for local androgens ("neuroandrogens") in the mesocorticolimbic system. Steroidogenic enzymes are expressed in mesocorticolimbic regions. Furthermore, following long-term gonadectomy, testosterone is nondetectable in the blood but detectable in the mesocorticolimbic system, using liquid chromatography tandem mass spectrometry. However, the physiological relevance of neuroandrogens remains unknown. Fourth, we review how anabolic-androgenic steroids (AAS) influence the mesocorticolimbic system. Fifth, we describe how androgens modulate the neurochemistry and structure of the mesocorticolimbic system, particularly with regard to dopaminergic signaling. Finally, we discuss evidence that androgens influence executive functions, including the effects of androgen deprivation therapy and AAS. Taken together, the evidence indicates that androgens are critical modulators of executive function. Similar to dopamine signaling, there might be optimal levels of androgen signaling within the mesocorticolimbic system for executive functioning. Future studies should examine the regulation and functions of neurosteroids in the mesocorticolimbic system, as well as the potential deleterious and enduring effects of AAS use.

Anabolic-androgenic steroids (AAS) increase sensitivity to uncertainty by inhibition of dopamine D1 and D2 receptors.

BACKGROUND: Anabolic-androgenic steroid abuse is implicated in maladaptive behaviors such as impaired cognition in humans. In a rat model, our lab has shown that testosterone decreases preference for a large/uncertain reward in probability discounting. Other studies have shown that androgens decrease dopamine D1 and D2 receptors in the nucleus accumbens shell, a region important for decision-making behavior in probability discounting. Thus, we attempted to restore selection of the large/uncertain reward in testosterone-treated rats by administering the D2 receptor agonist quinpirole or the D1 receptor agonist SKF81297 and testing probability discounting. METHODS: Adolescent male Long-Evans rats were treated chronically with high-dose testosterone (7.5 mg/kg) or vehicle (13% cyclodextrin in water), and tested for probability discounting after injections of saline, 0.1 and 0.5 mg/kg of quinpirole or SKF81297. Rats chose between a small/certain reward (1 sugar pellet, 100% probability) and a large/uncertain reward (4 pellets, decreasing probability: 100, 75, 50, 25, 0%). RESULTS: Testosterone-treated rats selected the large/uncertain reward significantly less than vehicle-treated controls after saline injection. However, acute injection with 0.1 mg/kg quinpirole increased large/uncertain reward preference in testosterone-treated rats only, indicated by a testosterone × quinpirole interaction. At 0.5 mg/kg, quinpirole increased large/uncertain reward preference in all rats. Acute injection with SKF81297 at 0.1 or 0.5 mg/kg rescued large/uncertain reward preference in testosterone-treated rats by eliminating the difference between groups. CONCLUSIONS: It appears that altered probability discounting behavior in testosterone-treated rats is due to both decreased D1 and D2 receptor function.

Expression and localization of myosin VI in developing mouse spermatids.

Myosin VI (MVI) is a versatile actin-based motor protein that has been implicated in a variety of different cellular processes, including endo- and exocytic vesicle trafficking, Golgi morphology, and actin structure stabilization. A role for MVI in crucial actin-based processes involved in sperm maturation was demonstrated in Drosophila. Because of the prominence and importance of actin structures in mammalian spermiogenesis, we investigated whether MVI was associated with actin-mediated maturation events in mammals. Both immunofluorescence and ultrastructural analyses using immunogold labeling showed that MVI was strongly linked with key structures involved in sperm development and maturation. During the early stage of spermiogenesis, MVI is associated with the Golgi and with coated and uncoated vesicles, which fuse to form the acrosome. Later, as the acrosome spreads to form a cap covering the sperm nucleus, MVI is localized to the acroplaxome, an actin-rich structure that anchors the acrosome to the nucleus. Finally, during the elongation/maturation phase, MVI is associated with the actin-rich structures involved in nuclear shaping: the acroplaxome, manchette, and Sertoli cell actin hoops. Since this is the first report of MVI expression and localization during mouse spermiogenesis and MVI partners in developing sperm have not yet been identified, we discuss some probable roles for MVI in this process. During early stages, MVI is hypothesized to play a role in Golgi morphology and function as well as in actin dynamics regulation important for attachment of developing acrosome to the nuclear envelope. Next, the protein might also play anchoring roles to help generate forces needed for spermatid head elongation. Moreover, association of MVI with actin that accumulates in the Sertoli cell ectoplasmic specialization and other actin structures in surrounding cells suggests additional MVI functions in spermatid movement across the seminiferous epithelium and in sperm release.

Sex differences and hormonal modulation of ethanol-enhanced risk taking in rats.

BACKGROUND: Ethanol (EtOH) intake correlates with increased risk-taking, and sex differences exist in both EtOH use and risk-taking in humans and rats. However, the interaction of sex and gonadal hormones to affect risk-taking under the influence of EtOH has not been determined. This was the focus of the current study. METHODS: Adult Long-Evans rats (n=18 males and females) were gonadectomized and received hormone replacement at physiologic levels or blank implants (n=7-9/group). Risk-taking was assessed with probability discounting, requiring rats to choose between a small/certain reward and a large/uncertain reward delivered with decreasing probability throughout each daily session. Before testing, rats received saline or EtOH (0.5 or 1.0g/kg) ip. RESULTS: In males, EtOH increased preference for the large/uncertain reward lever (F2,28=10.462, p<0.05). However, there was no effect of EtOH on lever preference in females (F1,30=0.914, p>0.05). At baseline, ORCHX+T males showed a greater preference for the large/uncertain reward lever then ORCHX males (F1,14=13.805, p<0.05). In females only, EtOH decreased choice latency relative to baseline (F1,10=7.25, p<0.05). EtOH decreased loss sensitivity in both sexes, with all rats exhibiting decreased lose-shift ratios (males: F2,18=5.10, p<0.05; females F2,10=4.37, p<0.05). CONCLUSIONS: These results show that EtOH, sex, and hormones interact to influence decision making. EtOH increases risk taking in males, but not in females. However, EtOH selectively decreases choice latency in females, and decreases loss sensitivity in both sexes. These findings are relevant to understanding human behavior, particularly in adolescents who experience increased hormone levels and often drink EtOH and engage in risky behavior.

The PULSE Vision & Change Rubrics, Version 1.0: A Valid and Equitable Tool to Measure Transformation of Life Sciences Departments at All Institution Types.

The PULSE Vision & Change Rubrics, version 1.0, assess life sciences departments' progress toward implementation of the principles of the Vision and Change report. This paper reports on the development of the rubrics, their validation, and their reliability in measuring departmental change aligned with the Vision and Change recommendations. The rubrics assess 66 different criteria across five areas: Curriculum Alignment, Assessment, Faculty Practice/Faculty Support, Infrastructure, and Climate for Change. The results from this work demonstrate the rubrics can be used to evaluate departmental transformation equitably across institution types and represent baseline data about the adoption of the Vision and Change recommendations by life sciences programs across the United States. While all institution types have made progress, liberal arts institutions are farther along in implementing these recommendations. Generally, institutions earned the highest scores on the Curriculum Alignment rubric and the lowest scores on the Assessment rubric. The results of this study clearly indicate that the Vision & Change Rubrics, version 1.0, are valid and equitable and can track long-term progress of the transformation of life sciences departments. In addition, four of the five rubrics have broad applicability and can be used to evaluate departmental transformation by other science, technology, engineering, and mathematics disciplines.

A pre-embedding immunogold approach reveals localization of myosin VI at the ultrastructural level in the actin cones that mediate Drosophila spermatid individualization.

Stable actin structures play important roles in the development and specialization of differentiated cells. How these structures form, are organized, and are used to mediate physiological processes is not well understood in most cases. In Drosophila testis, stable actin structures, called actin cones, mediate spermatid individualization, a large-scale cellular remodeling process. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. Myosin VI is an important player in proper actin cone organization and function. Myosin VI localizes to the cones' fronts and its specific localization is required for proper actin cone formation and function during individualization. To understand how these structures are organized and assembled, ultrastructural studies are important to reveal both organization of actin and the precise localization of actin regulators relative to regions with different filament organizations. In the present work, we have developed a novel pre-embedding immunogold-silver labeling method for high-resolution analysis of protein distribution in actin structures which allowed both satisfactory antibody labeling and good ultrastructural preservation. Electron microscopic studies revealed that myosin VI accumulated at the extreme leading edge of the actin cone and preferentially localized throughout the front meshwork of the cone where branched actin filaments were most concentrated. No myosin VI labeling was found adjacent to the membranes along the length of the cone or connecting neighboring cones. This method has potential to reveal important information about precise relationships between actin-binding proteins, membranes, and different types of actin structures.

Myosin VI regulates actin structure specialization through conserved cargo-binding domain sites.

Actin structures are often stable, remaining unchanged in organization for the lifetime of a differentiated cell. Little is known about stable actin structure formation, organization, or maintenance. During Drosophila spermatid individualization, long-lived actin cones mediate cellular remodeling. Myosin VI is necessary for building the dense meshwork at the cones' fronts. We test several ideas for myosin VI's mechanism of action using domain deletions or site-specific mutations of myosin VI. The head (motor) and globular tail (cargo-binding) domains were both needed for localization at the cone front and dense meshwork formation. Several conserved partner-binding sites in the globular tail previously identified in vertebrate myosin VI were critical for function in cones. Localization and promotion of proper actin organization were separable properties of myosin VI. A vertebrate myosin VI was able to localize and function, indicating that functional properties are conserved. Our data eliminate several models for myosin VI's mechanism of action and suggest its role is controlling organization and action of actin assembly regulators through interactions at conserved sites. The Drosophila orthologues of interaction partners previously identified for vertebrate myosin VI are likely not required, indicating novel partners mediate this effect. These data demonstrate that generating an organized and functional actin structure in this cell requires multiple activities coordinated by myosin VI.

Coiled-coil-mediated dimerization is not required for myosin VI to stabilize actin during spermatid individualization in Drosophila melanogaster.

Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.

Genetic characterization of the Drosophila jaguar322 mutant reveals that complete myosin VI loss of function is not lethal.

Myosin VI is an actin-based motor that has been implicated in many cellular processes. Studies in vertebrates have demonstrated that animals lacking this ubiquitously expressed myosin are viable. However in Drosophila, myosin VI loss of function has been thought to be lethal. We show here that complete loss of myosin VI is not lethal in flies and that the previously reported lethality of the null mutation (jar322) is most likely due to deletion of a neighboring gene. Maternally provided myosin VI does not account for the survival of myosin VI null animals. Mutant animals are recovered at a lower than expected Mendelian frequency, suggesting that myosin VI participates in processes which contribute to normal development, but its participation is not essential.

Proper cellular reorganization during Drosophila spermatid individualization depends on actin structures composed of two domains, bundles and meshwork, that are differentially regulated and have different functions.

During spermatid individualization in Drosophila, actin structures (cones) mediate cellular remodeling that separates the syncytial spermatids into individual cells. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. We show here that the two domains form separately in time, are regulated by different sets of actin-associated proteins, can be formed independently, and have different roles. Newly forming cones were composed only of bundles, whereas the meshwork formed later, coincident with the onset of cone movement. Polarized distributions of myosin VI, Arp2/3 complex, and the actin-bundling proteins, singed (fascin) and quail (villin), occurred when movement initiated. When the Arp2/3 complex was absent, meshwork formation was compromised, but surprisingly, the cones still moved. Despite the fact that the cones moved, membrane reorganization and cytoplasmic exclusion were abnormal and individualization failed. In contrast, when profilin, a regulator of actin assembly, was absent, bundle formation was greatly reduced. The meshwork still formed, but no movement occurred. Analysis of this actin structure's formation and participation in cellular reorganization provides insight into how the mechanisms used in cell motility are modified to mediate motile processes within specialized cells.

Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development.

Drosophila melanogaster bristle development is dependent on actin assembly, and prominent actin bundles form against the elongating cell membrane, giving the adult bristle its characteristic grooved pattern. Previous work has demonstrated that several actin-regulating proteins are required to generate normal actin bundles. Here we have addressed how two actin regulators, capping protein, a barbed end binding protein, and the Arp2/3 complex, a potent actin assembly nucleator, function to generate properly organized bundles. As predicted from studies in motile cells, we find that capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, these proteins do not primarily act directly on bundles, but rather on a dynamic population of actin filaments that are not part of the bundles. These nonbundle filaments, termed snarls, play an important role in determining the number and spacing of the actin bundles. Reduction of capping protein leads to an increase in snarls, which prevents actin bundles from properly attaching to the membrane. Conversely, loss of an Arp2/3 complex component leads to a loss of snarls and accumulation of excess membrane-attached bundles. These results indicate that in nonmotile cells dynamic actin filaments can function to regulate the positioning of stable actin structures. In addition, our results suggest that the Arpc1 subunit may have an additional function, independent of the rest of the Arp2/3 complex.

Androcam is a tissue-specific light chain for myosin VI in the Drosophila testis.

Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue.

Myosin VI stabilizes an actin network during Drosophila spermatid individualization.

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.

Myosin VI: a structural role in actin organization important for protein and organelle localization and trafficking.

Myosin VI is a member of a superfamily of actin-based motors with at least 18 different sub-types or classes. Myosins are best known as proteins that use ATP-hydrolysis-mediated conformational changes to move along actin filaments. Because of this property, some myosins, including myosins I, V, and VI, are thought to be transporters of vesicle or protein cargoes. Myosin VI has been implicated in many seemingly different processes through functional studies in flies, worms and mammals. In several cases, its role is not easily explained by transport along actin. In addition, some of the biochemical and biophysical properties of myosin VI suggest other mechanisms of action. In this review, we summarize recent data that suggest diverse functions for myosin VI and offer an explanation for how myosin VI may function similarly in all of them. We hypothesize that the main function of myosin VI is to bind tightly to actin, stabilizing actin cytoskeletal structures and linking actin structures to membranes and protein complexes.

Converting a motor to an anchor.

Myosin VI can move along actin filaments to serve as a transport motor. It is also thought to anchor vesicles or proteins to actin. How these two diverse activities, which require very different modes of interaction with actin, are mediated is not understood. Using single molecule observations, Altman et al. (2004 [this issue of Cell]) demonstrate that load applied to myosin VI can convert this motor from a transporter to an anchor.