Structural characterization of cytoskeleton regulating protein villin and its C-terminal modular domains

Villin is a modular protein that regulates F-actin bundles in the microvilli of absorptive epithelial cells in the intestine. At low (10-100 nM) calcium levels, Villin is an F-actin bundling agent supporting the specialized brush border membrane of the absorptive epithelium. At intermediate micromolar calcium levels, Villin nucleates and caps the barbed ends of F-actin and in high (\u3e 100 μM) calcium Villin is an F-actin severing agent (Bretsher & Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al. 1980). The amino acid sequence of Villin has seven modular domains. The first six Villin domains (D1-D6) form a core of ~50% sequence identity with Gelsolin; and contain a Ca2+-dependent actin-binding site associated with the D1-D3 fragment. The last domain, Villin\u27s unique C-terminal headpiece (HP), contains the other F-actin binding site, which is Ca2+-independent (Bretsher & Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al. 1980). Recent investigation by Nuclear Magnetic Resonance (NMR) Spectroscopy and Negative-Stain Electron Microscopy (EM) of the backbone dynamics and actin-binding of Villin\u27s D6-HP, 208-residue, C-terminal modular fragment, revealed that: a) folded domains D6 and HP are interacting only via a largely unfolded 40-residue linker, and b) at millimolar calcium levels, the monomeric D6-HP fragment bundles F-actin and has two actin binding sites; one, which is previously known on HP, and the other is novel, cryptic and Ca2+-dependent, associated with domain D6 or the linker (Smirnov et al., 2007). We have investigated how the domain structure, domain-domain and linker-domain interactions in D6-HP fragment of Villin define its actin regulation properties. Toward this goal, we are: a) making the D6 and D6-HP NMR samples; b) determining the NMR resonance assignment of isolated D6; and c) elucidating the solution structure of D6 domain in isolation and within the D6-HP fragment. Our NMR data indicate that the D6 protein fragment in isolation likely adopts a Gelsolin-like fold and that HP and D6 structures in isolation resemble those in the context of the larger modular fragment D6-HP. The potential effect of the linker on the D6 and HP domains structure is exemplified by the noticeable chemical shift differences for residue 84 of D6 and residue 166 of HP (15N-HSQC spectrum of D6-HP vs. D6 and HP in isolation). These two positions are ~23 residues away from either end of the linker and located on the surface of these domains. In the absence of calcium, Gelsolin adopts a compact, inactive conformation stabilized by the 12-residue C-terminal helix. This helix was suggested to keep together Gelsolin domains D2 and D6 as a latch closed in low calcium and released at higher calcium levels (Robinson et al., 1999). Our ensuing structural study of D6-HP will clarify whether the linker sequence in D6-HP corresponding to this C-terminal helix of Gelsolin forms a helix as well and thus may or not undergo a gelsolin-like, calcium-induced rearrangement. The solution structure of D6 will be determined by NMR and analyzed in combination with the complete solution structure of HP and known structural properties of D6-HP. Together with the calcium and F-actin binding properties of D6 in isolation (currently under study), these data will clarify the role of the C-terminal domains of Villin in its activity as a physiologically principal actin regulator of microvilli

Emotion and Warmth Modulation in Women Leaders: A Qualitative Exploratory Study

Gender stereotypes dictate that women are and should be warm, whereas men are and should be competent. While prior work has explored how women manage stereotypic expectations about their competence, there is less research on the lived experiences of women leaders navigating the warmth dimension of these stereotypes. This qualitative study initially explored the possibility that women leaders may modulate emotional displays in service of conveying warmth. The research questions evolved over time and the study ultimately aimed to understand the following research questions: (1) Do women leaders in male-typed jobs modulate emotional displays in the workplace? If so, why, how, and what are the outcomes? (2) Do women leaders in male-typed jobs modulate warmth displays in the workplace? If so, why, how, and what are the outcomes? The study included data from semi-structured interviews with 22 women leaders in male-typed contexts. The data ultimately revealed that the vast majority of participants engaged in emotion modulation at work. There were a variety of reasons underlying this process, including participants viewing modulating emotions as a component of their competence in their roles, using emotionality as a tool in the workplace, and noting that specific emotions were unacceptable to express in their workplace. They also identified how they managed their emotions, reporting strategies that ranged from within the workplace to outside the workplace, as well as intrapersonal versus interpersonal strategies. Finally, they reported the mostly negative intrapersonal outcomes of modulating emotions, including feelings of fatigue and inauthenticity. With regard to warmth, the majority of participants reported modulating warmth at work. Participants modulated warmth for various reasons, including viewing warmth as a component of leadership, in response to others’ gendered expectations for warmth displays, and reflecting on actual or predicted outcomes of warmth displays to guide subsequent warmth displays. They conveyed warmth in a variety of ways, such as appearing friendly and approachable, resolving conflict with others, and creating a supportive team environment. Finally, they reported myriad outcomes associated with warmth modulation, including fatigue and discomfort, as well as warmth displays reducing credibility or a failure to display warmth resulting in negative professional outcomes. One final theme also emerged, bridging across warmth and emotionality. At times, participants suppressed negative emotions, then amplified warmth behaviors. They also displayed positive emotions, then amplified subsequent warmth behaviors. The findings suggested that women leaders may be modulating both emotions and warmth independently of one another, yet there are also instances where warmth modulation directly follows emotional modulation. This study provides compelling evidence that women leaders engage in labor, outside of explicit role responsibilities, in managing both emotions and warmth in the workplace. Given the depth and complexity of the findings as well as the limitations of this study, additional research is required to replicate these findings with other methodological approaches, designs, and samples. The results point to several theoretical areas that may benefit from greater refinement and differentiation, including the relationship between emotional modulation and warmth modulation. Finally, there are numerous implications for practice at the organization, group, and individual levels

Female rat sexual behavior is unaffected by perinatal fluoxetine exposure

Serotonin plays an important role in adult female sexual behavior, however little is known about the influence of serotonin during early development on sexual functioning in adulthood. During early development, serotonin acts as neurotrophic factor, while it functions as a modulatory neurotransmitter in adulthood. The occurrence of serotonin release, could thus have different effects on behavioral outcomes, depending on the developmental period in which serotonin is released. Because serotonin is involved in the development of the HPG axis which is required for puberty establishment, serotonin could also alter expression patterns of for instance the estrogen receptor ɑ (ERɑ). The aim of our study was to investigate the effects of increased serotonin levels during early development on adult female rat sexual behavior during the full behavioral estrus in a seminatural environment. To do so, rats were perinatally exposed with the selective serotonin reuptake inhibitor (SSRI) fluoxetine (10 mg/kg FLX) and sexual performance was tested during adulthood. All facets of female sexual behavior between the first and last lordosis (behavioral estrus), and within each copulation bout of the behavioral estrus were analyzed. Besides the length and onset of the behavioral estrus and the sexual behaviors patterns, other social and conflict behavior were also investigated. In addition, we studied the effects of perinatal FLX exposure on ERɑ expression patterns in the medial preoptic nucleus, ventromedial nucleus of the hypothalamus, medial amygdala, bed nucleus of the stria terminalis, and the dorsal raphé nucleus. The results showed that perinatal fluoxetine exposure has no effect on adult female sexual behavior. The behavioral estrus of FLX-females had the same length and pattern as CTR-females. In addition, FLX- and CTR-females showed the same amount of paracopulatory behavior and lordosis, both during the full behavioral estrus and the "most active bout". Furthermore, no differences were found in the display of social and conflict behaviors, nor in ERɑ expression patterns in the brain. We conclude that increases in serotonin levels during early development do not have long-term consequences for female sexual behavior in adulthood.</p

Gray matter volume in schizophrenia and bipolar disorder with psychotic features

There is growing evidence that schizophrenia (SZ) and bipolar disorder (BD) overlap significantly in risk factors, neurobiological features, clinical presentations, and outcomes. SZ is characterized by well documented gray matter (GM) abnormalities in multiple frontal, temporal and subcortical structures. Recent voxel-based morphometry (VBM) studies and meta-analyses in BD also report GM reductions in overlapping, albeit less widespread, brain regions. Psychosis, a hallmark of SZ, is also experienced by a significant proportion of BD patients and there is evidence that psychotic BD may be characterized by specific clinical and pathophysiological features. However, there are few studies comparing GM between SZ and psychotic BD. In this study we compared GM volumes in a sample of 58 SZ patients, 28 BD patients experiencing psychotic symptoms and 43 healthy controls using whole-brain voxel-based morphometry. SZ patients had GM reductions in multiple frontal and temporal regions compared to healthy controls and in the subgenual cortex compared to psychotic BD patients. GM volume was increased in the right posterior cerebellum in SZ patients compared to controls. However, psychotic BD patients did not show significant GM deficits compared to healthy controls or SZ patients. We conclude that GM abnormality as measured by VBM analysis is less pronounced in psychotic BD compared to SZ. This may be due to disease-specific factors or medications used more commonly in BD. (C) 2012 Elsevier B.V. All rights reserved

Gelsolin-like Activation of Villin: Calcium Sensitivity of the Long Helix in Domain 6

Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1–V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villin’s functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-<i>K</i>_d calcium binding in V6 (<i>K</i>_d1 of 22 μM and <i>K</i>_d2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism

Gelsolin-like Activation of Villin: Calcium Sensitivity of the Long Helix in Domain 6

Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1–V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villin’s functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-<i>K</i>_d calcium binding in V6 (<i>K</i>_d1 of 22 μM and <i>K</i>_d2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism

Gelsolin-like Activation of Villin: Calcium Sensitivity of the Long Helix in Domain 6

Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1–V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villin’s functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-<i>K</i>_d calcium binding in V6 (<i>K</i>_d1 of 22 μM and <i>K</i>_d2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism