Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. ...Recently, we developed a novel actin probe, β‐actin‐EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP‐β‐actin fusion protein that over‐assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam‐like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact‐EGFP or anti‐actin antibodies. In addition, immunofluorescence staining and expression of cofilin‐EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin‐negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.
Abstract The existence of individual differences in personality can be puzzling from an evolutionary perspective. This paper offers a general framework for addressing this puzzle by combining ...insights from evolutionary, situational, and personality perspectives. To arrive at this framework, we first discuss three key evolutionary models for explaining personality variation: (1) selective neutrality, (2) mutation-selection balance, and (3) balancing selection. Second, we review four models of personality: (1) the General Factor of Personality, (2) The Big Two, (3) the Big Five, and (4) the six-dimensional HEXACO model. Third, we use situational affordances and trait activation perspectives to offer an integrative model of HEXACO domain-specific situational affordances. Finally, we use these perspectives to provide 18 propositions about situation, trait, and outcome activation (STOA) mechanisms which may help explain the maintenance of individual differences in six dimensions of personality.
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•HIV-1 Gag protein adopts a compact (C-Gag) form in cells.•C-Gag formation requires both MA and NC RNA-binding domains.•C-Gag is stabilized by an intramolecular interaction between MA ...and CA domains.•Mutation altering C-Gag formation reduce infectious pseudoparticles production and infectivity.
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
Tyrosine kinase 2 (TYK2) associates with interferon (IFN) alpha receptor, IL‐10 receptor (IL‐10R) beta and other cytokine receptor subunits for signal transduction, in response to various cytokines, ...including type‐I and type‐III IFNs, IL‐6, IL‐10, IL‐12 and IL‐23. Data on TYK2 dependence on cytokine responses and in vivo consequences of TYK2 deficiency are inconsistent. We investigated a TYK2 deficient patient, presenting with eczema, skin abscesses, respiratory infections and IgE levels >1000 U/mL, without viral or mycobacterial infections and a corresponding cellular model to analyze the role of TYK2 in type‐III IFN mediated responses and NK‐cell function. We established a novel simple diagnostic monocyte assay to show that the mutation completely abolishes the IFN‐α mediated antiviral response. It also partly reduces IL‐10 but not IL‐6 mediated signaling associated with reduced IL‐10Rβ expression. However, we found almost normal type‐III IFN signaling associated with minimal impairment of virus control in a TYK2 deficient human cell line. Contrary to observations in TYK2 deficient mice, NK‐cell phenotype and function, including IL‐12/IL‐18 mediated responses, were normal in the patient. Thus, preserved type‐III IFN responses and normal NK‐cell function may contribute to antiviral protection in TYK2 deficiency leading to a surprisingly mild human phenotype.
IFN‐λ mediated signal transduction is functionally retained in TYK2 deficient human cell lines. This, together with functional NK cell responses in vitro, might account for the absence of severe viral infections in patients with TYK2 deficiency despite the lack of IFN‐α mediated signal transduction.
During molecular cloning, screening bacterial transformants is a time-consuming and labor-intensive process; however, tractable tools that can be applied to various vectors for visual confirmation of ...desired colonies are limited. Recently, we reported that translational enhancement by a
gene sequence (TED) boosted protein expression even without an expression inducer in
. Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein enhanced by TED. By inserting a module related to TED into the cloning site in advance, we effectively screened
colonies harboring the desired plasmid functions in a prokaryote (
) or eukaryote (
). Thus, our system represents a user-friendly technique for cloning.
This work presents a simple, reliable and cost-effective bacterial colony screening method using green fluorescent protein (GFP) expression. No expression inducer is required for GFP expression. In addition, this method is easily applied to any vector by insertion of a module related to GFP expression.
SUMMARY
Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An ...increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant–pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)‐GFP vector and Agrobacterium‐mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV‐GFP system, we initially tested it with well‐described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV‐GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV‐GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV‐GFP system in identifying apoplastic effectors. The easy operation, time‐saving nature, broad effectiveness, and low technical requirements of the TMV‐GFP system make it a promising approach for high‐throughput screening of effectors with immune interference activity from various pathogens.
Significance Statement
The development of a robust high‐throughput functional screening assay for pathogen effectors will facilitate the study in plant‐pathogen interactions. We established a novel screening system using the TMV‐GFP vector, enabling the rapid identification of effectors that interfere with plant immunity by observing GFP fluorescence under a UV lamp within a few days. The TMV‐GFP system is a promising approach for high‐throughput screening of effectors from diverse pathogens.
Neuropathic pain is pain caused by damage to the somatosensory nervous system. Both degenerating injured nerves and neighboring sprouting nerves can contribute to neuropathic pain. However, the ...mesoscale changes in cutaneous nerve fibers over time after the loss of the parent nerve has not been investigated in detail. In this study, we followed the changes in nerve fibers longitudinally in the toe tips of mice that had undergone spared nerve injury (SNI). Nav1.8‐tdTomato, Thy1‐GFP and MrgD‐GFP mice were used to observe the small and large cutaneous nerve fibers. We found that peripheral nerve plexuses degenerated within 3 days of nerve injury, and free nerve endings in the epidermis degenerated within 2 days. The timing of degeneration paralleled the initiation of mechanical hypersensitivity. We also found that some of the Nav1.8‐positive nerve plexuses and free nerve endings in the fifth toe survived, and sprouting occurred mostly from 7 to 28 days. The timing of the sprouting of nerve fibers in the fifth toe paralleled the maintenance phase of mechanical hypersensitivity. Our results support the hypotheses that both injured and intact nerve fibers participate in neuropathic pain, and that, specifically, nerve degeneration is related to the initiation of evoked pain and nerve sprouting is related to the maintenance of evoked pain.
We have developed a method for the longitudinal intravital observation of changes in peripheral nerve fibers. This method provides a high‐resolution approach for investigating epidermal and dermal nerve terminals as well as nerve plexuses and were extremely stable for at least 28 days. Using the SNI model, we found that the degeneration of the peripheral nerve terminals and plexuses over 2–3 days was aligned closely with the initiation of mechanical hypersensitivity. In addition, we obtained clear images of sprouting in the lateral part of the paw, which may be related to the maintenance of neuropathic pain symptoms.
To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy‐inducing potential of a panel of fatty acids ...in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate‐induced, but not oleate‐induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non‐canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.
Synopsis
A systematic screen in cancer cells reveals that unsaturated and saturated fatty acids induce autophagy via distinct pathways, with unsaturated fatty acids acting in a Golgi‐dependent but Beclin‐1‐independent manner.
Saturated and unsaturated fatty acids promote autophagy, in vitro and in vivo, via different molecular mechanisms.
The saturated fatty acid palmitate stimulates canonical, BECN1‐ and PIK3C3‐dependent autophagic responses that involve JNK1, PKR and AMPK.
The unsaturated fatty acid oleate promotes a non‐canonical BECN1‐independent autophagic response that requires an intact Golgi apparatus.
Oleate‐induced non‐canonical autophagy is conserved in human cells, mice, yeast and nematodes.
A systematic screen in cancer cells reveals that unsaturated and saturated fatty acids induce autophagy via distinct pathways, with unsaturated fatty acids acting in a Golgi‐dependent but Beclin‐1‐independent manner.
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