Light sheet fluorescence microscopy using axi-symmetric binary phase filters.

Light sheet fluorescence microscopy (LSFM) has become an indispensable tool in biomedical studies owing to its depth-sectioning capability and low photo-bleaching. The axial resolution in LSFM is determined mainly by the thickness of the illumination sheet, and a high numerical-aperture lens is thus preferred in the illumination to increase the axial resolution. However, a rapid divergence of the illumination beam limits the effective field-of-view (FoV), that provides high-resolution images. Several strategies have been demonstrated for FoV enhancement, which involve the use of Bessel or Airy beams, for example. However, the generation of these beams requires complicated optical setup or phase filters with continuous phase distributions, which are difficult to manufacture. In contrast, a binary phase filter (BPF) comprising concentric rings with 0 or π phases produces a response similar to its continuous original and is easy to realize. Here, we present a novel form of LSFM that integrates BPFs derived from two representative axi-symmetric aberrations, including phase axicon and spherical aberrations, to improve the imaging performance. We demonstrate that these BPFs significantly increase the FoV, and those derived from axicon generate self-reconstructing beams, which are highly desirable in imaging through scattering specimens. We validate its high-contrast imaging capability over extended FoV by presenting three-dimensional images of microspheres, imaginal disc of Drosophila larva, and Arabidopsis.

BRPF3-HUWE1-mediated regulation of MYST2 is required for differentiation and cell-cycle progression in embryonic stem cells.

Brpf-histone acetyltransferase (HAT) complexes have important roles in embryonic development and regulating differentiation in ESCs. Among Brpf family, Brpf3 is a scaffold protein of Myst2 histone acetyltransferase complex that plays crucial roles in gene regulation, DNA replication, development as well as maintaining pluripotency in embryonic stem cells (ESCs). However, its biological functions in ESCs are not elucidated. In this study, we find out that Brpf3 protein level is critical for Myst2 stability and E3 ligase Huwe1 functions as a novel negative regulator of Myst2 via ubiquitin-mediated degradation. Importantly, Brpf3 plays an antagonistic role in Huwe1-mediated degradation of Myst2, suggesting that protein-protein interaction between Brpf3 and Myst2 is required for retaining Myst2 stability. Further, Brpf3 overexpression causes the aberrant upregulation of Myst2 protein levels which in turn induces the dysregulated cell-cycle progression and also delay of early embryonic development processes such as embryoid-body formation and lineage commitment of mouse ESCs. The Brpf3 overexpression-induced phenotypes can be reverted by Huwe1 overexpression. Together, these results may provide novel insights into understanding the functions of Brpf3 in proper differentiation as well as cell-cycle progression of ESCs via regulation of Myst2 stability by obstructing Huwe1-mediated ubiquitination. In addition, we suggest that this is a useful report which sheds light on the function of an unknown gene in ESC field.

Necrotic cell death induces melanotic mass formation in Drosophila.

The immune system protects its host from not only invading parasites and parasitoids, but also altered self tissue, including dying cells. Necrotic cells are strongly immunogenic, but in Drosophila this has not been directly addressed, due partially to the fact that knowledge about necrosis in Drosophila currently lags behind that for other models. Upon the loss of cell matrix attachment, endocycling polyploid tissues of the Drosophila larva undergo autophagy instead of apoptosis; we employed this system as a model to examine cell death modalities and immunity. Here, we report that larval fat body cells depleted of integrin undergo not only autophagy, but also necrotic cell death, and that a blockade of reaper, grim, hid, or the downstream caspases enhances necrosis. These cells elicit melanotic mass formation, an autoimmune-like response. We also show that necrosis is the main cause of melanotic mass formation in these anchorage-depleted polyploid cells.

Interplay between integrins and PI4P5K Sktl is crucial for cell polarization and reepithelialisation during Drosophila wound healing.

Phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P2] regulates cell adhesion and actin dynamics during cell migration. PI(4,5)P2 binds various components of the cell adhesion machinery, but how these processes affect migration of the epithelial cell sheet is not well understood. Here, we report that PI(4,5)P2 and Sktl, the kinase that converts PI4P to PI(4,5)P2, are both localized to the rear side of cells during wound healing of the Drosophila larval epidermis. The Sktl localization requires JNK pathway activation and integrins, but not PVR. The sktl knockdown epidermis displays strong defects in would closure, reminiscent of the JNK-depleted epidermis, and shows severe disruption of cell polarity, as determined by myosin II localization. Sktl and ßPS integrin colocalize at the rear side of cells forming the trailing edge during wound healing and the two are inter-dependent in that the absence of one severely disrupts the rear localization of the other. These results strongly suggest that the JNK pathway regulates the rear localization of Sktl and integrins and the interplay between Sktl and integrins sets up cell polarity, which is crucial for reepithelialisation during wound healing.

cdc37 is essential for JNK pathway activation and wound closure in Drosophila.

Wound closure in the Drosophila larval epidermis mainly involves nonproliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. We discovered that both cell growth and migration in Drosophila require the cochaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin ß subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin ß during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing.

Inflammatory production of reactive oxygen species by Drosophila hemocytes activates cellular immune defenses.

The production of reactive oxygen species (ROS) is a prominent response to infection among innate immune cells such as macrophages and neutrophils. To better understand the relationship between antimicrobial and regulatory functions of blood cell ROS, we have characterized the ROS response to infection in Drosophila hemocytes. Using fluorescent probes, we find a biphasic hemocyte ROS response to bacterial infection. In the first hour, virtually all hemocytes generate a transient ROS signal, with nonphagocytic cells including prohemocytes and crystal cells displaying exceptionally strong responses. A distinct, and more delayed ROS response starting at 90 min is primarily within cells that have engulfed bacteria, and is sustained for several hours. The early response has a clear regulatory function, as dampening or intensifying the intracellular ROS level has profound effects on plasmatocyte activation. In addition, ROS are necessary and sufficient to activate JNK signalling in crystal cells, and to promote JNK-dependent crystal cell rupture. These findings indicate that Drosophila will be a promising model in which to dissect the mechanisms of ROS stimulation of immune activation.

Requirement for and polarized localization of integrin proteins during Drosophila wound closure.

Wound reepithelialization is an evolutionarily conserved process in which skin cells migrate as sheets to heal the breach and is critical to prevent infection but impaired in chronic wounds. Integrin heterodimers mediate attachment between epithelia and underlying extracellular matrix and also act in large signaling complexes. The complexity of the mammalian wound environment and evident redundancy among integrins has impeded determination of their specific contributions to reepithelialization. Taking advantage of the genetic tools and smaller number of integrins in Drosophila, we undertook a systematic in vivo analysis of integrin requirements in the reepithelialization of skin wounds in the larva. We identify αPS2-ßPS and αPS3-ßPS as the crucial integrin dimers and talin as the only integrin adhesion component required for reepithelialization. The integrins rapidly accumulate in a JNK-dependent manner in a few rows of cells surrounding a wound. Intriguingly, the integrins localize to the distal margin in these cells, instead of the frontal or lamellipodial distribution expected for proteins providing traction and recruit nonmuscle myosin II to the same location. These findings indicate that signaling roles of integrins may be important for epithelial polarization around wounds and lay the groundwork for using Drosophila to better understand integrin contributions to reepithelialization.

Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing.

Cell-cell fusion is widely observed during development and disease, and imposes a dramatic change on participating cells. Cell fusion should be tightly controlled, but the underlying mechanism is poorly understood. Here, we found that the JAK/STAT pathway suppressed cell fusion during wound healing in the Drosophila larval epidermis, restricting cell fusion to the vicinity of the wound. In the absence of JAK/STAT signaling, a large syncytium containing a 3-fold higher number of nuclei than observed in wild-type tissue formed in wounded epidermis. The JAK/STAT ligand-encoding genes upd2 and upd3 were transcriptionally induced by wounding, and were required for suppressing excess cell fusion. JNK (also known as Basket in flies) was activated in the wound vicinity and activity peaked at ∼8 h after injury, whereas JAK/STAT signaling was activated in an adjoining concentric ring and activity peaked at a later stage. Cell fusion occurred primarily in the wound vicinity, where JAK/STAT activation was suppressed by fusion-inducing JNK signaling. JAK/STAT signaling was both necessary and sufficient for the induction of ßPS integrin (also known as Myospheroid) expression, suggesting that the suppression of cell fusion was mediated at least in part by integrin protein.

PINK1 phosphorylates transglutaminase 2 and blocks its proteasomal degradation.

Parkinson's disease (PD) is characterized by progressive dopaminergic neuronal loss and the formation of abnormal protein aggregates, referred to as Lewy bodies (LBs). PINK1 is a serine/threonine protein kinase that protects cells from stress-induced mitochondrial dysfunction. PINK1 gene mutations cause one form of autosomal recessive early-onset PD. Transglutaminase 2 (TG2) is an intracellular protein cross-linking enzyme that has an important role in LB formation during PD pathogenesis. This study identifies PINK1 as a novel TG2 binding partner and shows that PINK1 stabilizes the half-life of TG2 via inhibition of TG2 ubiquitination and subsequent proteasomal degradation. PINK1 affects TG2 stability in a kinase-dependent manner. In addition, PINK1 directly phosphorylates TG2 in carbonyl cyanide m-chlorophenyl hydrazine-induced mitochondrial damaged states, thereby enhancing TG2 accumulation and intracellular protein cross-linking products. This study further confirms the functional link between upstream PINK1 and downstream TG2 in Drosophila melanogaster. These data suggest that PINK1 positively regulates TG2 activity, which may be closely associated with aggresome formation in neuronal cells.

Basement membrane and cell integrity of self-tissues in maintaining Drosophila immunological tolerance.

The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. Here, we established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. We found that the basement membrane endows otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, we found that cell integrity, as determined by cell-cell interaction and apicobasal polarity, functions as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity.

Requirement for Pak3 in Rac1-induced organization of actin and myosin during Drosophila larval wound healing.

Rho-family small GTPases regulate epithelial cell sheet migration by organizing actin and myosin during wound healing. Here, we report that Pak3, but not Pak1, is a downstream target protein for Rac1 in wound closure of the Drosophila larval epidermis. Pak3-deficient larvae failed to close a wound hole and this defect was not rescued by Pak1 expression, indicating differential functions of the two proteins. Pak3 localized to the wound margin, which selectively required Rac1. Pak3-deficient larvae showed severe defects in actin-myosin organization at the wound margin and in submarginal cells, which was reminiscent of the phenotypes of Rac1-deficient larvae. These results suggest that Pak3 specifically mediates Rac1 signaling in organizing actin and myosin during Drosophila epidermal wound healing.

Crystal structure of Fushi tarazu factor 1 ligand binding domain/Fushi tarazu peptide complex identifies new class of nuclear receptors.

The interaction between the orphan nuclear receptor FTZ-F1 (Fushi tarazu factor 1) and the segmentation gene protein FTZ is critical for specifying alternate parasegments in the Drosophila embryo. Here, we have determined the structure of the FTZ-F1 ligand-binding domain (LBD)·FTZ peptide complex using x-ray crystallography. Strikingly, the ligand-binding pocket of the FTZ-F1 LBD is completely occupied by helix 6 (H6) of the receptor, whereas the cofactor FTZ binds the co-activator cleft site of the FTZ-F1 LBD. Our findings suggest that H6 is essential for transcriptional activity of FTZ-F1; this is further supported by data from mutagenesis and activity assays. These data suggest that FTZ-F1 might belong to a novel class of ligand-independent nuclear receptors. Our findings are intriguing given that the highly homologous human steroidogenic factor-1 and liver receptor homolog-1 LBDs exhibit sizable ligand-binding pockets occupied by putative ligand molecules.

Protein O-GlcNAcylation regulates Drosophila growth through the insulin signaling pathway.

Modification of nuclear and cytosolic proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) is ubiquitous in cells. The in vivo function of the protein O-GlcNAcylation, however, is not well understood. Here, we manipulated the cellular O-GlcNAcylation level in Drosophila and found that it promotes developmental growth by enhancing insulin signaling. This increase in growth is due mainly to cell growth and not to cell proliferation. Our data suggest that the increase in the insulin signaling activity is mediated, at least in part, through O-GlcNAcylation of Akt. These results indicate that O-GlcNAcylation is one of the crucial mechanisms involved in control of insulin signaling during Drosophila development.

Binding preference of p62 towards LC3-ll during dopaminergic neurotoxin-induced impairment of autophagic flux.

Accumulating evidence has revealed that autophagy may be beneficial for treatment of neurodegenerative diseases through removal of abnormal protein aggregates. However, the critical autophagic events during neurodegeneration remain to be elucidated. Here, we investigated whether prototypic autophagic events occur in the MN9D dopaminergic neuronal cell line upon exposure to N-methyl-4-phenylpyridinium (MPP (+) ), a well-known dopaminergic neurotoxin. MPP (+) treatment induced both morphological and biochemical characteristics of autophagy, such as accumulation of autophagic vacuoles and LC3-II form and decreased p62 levels. Further investigation revealed that these phenomena were largely the consequences of blocked autophagic flux. Following MPP (+) treatment, levels of LC3-II formed and p62 dramatically increased in the Triton X-100-insoluble fraction. Levels of ubiquitinated proteins also increased in this fraction. Further colocalization analyses revealed that the punctated spots positive for both p62 and LC3 were more intense following MPP (+) treatment, suggesting drug-induced enrichment of these two proteins in the insoluble fraction. Intriguingly, reciprocal immunoprecipitation analysis revealed that p62 mainly precipitated with LC3-II form following MPP (+) treatment. Transient transfection of the mutant form of Atg4B, Atg4B (C74A) , which inhibits LC3 processing, dramatically decreased binding between p62 and LC3-II form. Taken together, our results indicate that p62 can be efficiently localized to autophagic compartments via preferential binding with LC3-II form. This colocalization may assist in removal of detergent-insoluble forms of damaged cellular proteins during dopaminergic neurotoxin-induced impairment of autophagic flux.

Proteomic analysis of expression and protein interactions in a 6-hydroxydopamine-induced rat brain lesion model.

Parkinson's disease (PD) is the second most common neurodegenerative disorder caused by selective degeneration of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Although mitochondrial abnormality, oxidative stress and proteasomal dysfunction are recognized as major contributors to the progression of PD, there is a limited understanding of the key molecular events that provoke degeneration of DA neurons. Using a proteomic approach, we attempted to identify profiles of proteins with altered expression levels in rats following unilateral stereotaxic injection of 6-hydroxydopamine into the SNc. Protein expression profiles of these proteins in the substantia nigra and the striatum were made using two-dimensional gel electrophoresis in conjunction with a mass spectrometry. More than 70 identified proteins displayed significant differences in their temporal and spatial expression pattern between experimental and vehicle-operated control groups. Based on the identity of the proteins, we further searched for potential binding partners using biological databases available on the web and constructed a protein interaction network. Among several interconnected proteins in the network, we verified the interaction between prohibitin and the NADH-ubiquinone oxidoreductase 30kDa subunit (NDUFS3 subunit; a mitochondrial complex I subunit) by co-immunoprecipitation. We also confirmed, using immunohistochemical localization, that both prohibitin and the NDUFS3 subunit were increased in the dying DA neurons, suggesting its potential role in regulating mitochondrial function in dying DA neurons. Furthermore, knockdown of prohibitin accelerated 6-hydroxydopamine-induced cell death in SH-SY5Y cells. Our results raise the possibility that interconnected proteins in the network may positively or negatively impact the progression of DA neuronal death.

Rho-family small GTPases are required for cell polarization and directional sensing in Drosophila wound healing.

A wound induces cell polarization, in which myosin II is localized at the rear end of individual cells in a migrating epithelial sheet of the Drosophila larval epidermis. Here, we use myosin localization to demonstrate that Rac1, Cdc42, and Rho1 are each required for cell polarization and directional sensing of the wound. The three GTPases are also required for actin cable formation at the wound leading edge. Rac1, Cdc42, and Rho1 act upstream of c-Jun N-terminal kinase (JNK) to organize actin assembly. These results highlight the similarities between the molecular mechanism of Drosophila wound healing and those of Drosophila embryonic dorsal closure and the chemotactic response of Dictyostelium and leukocytes.

Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing.

We investigated cell shape changes during wound closure in the Drosophila larval epidermis. During reepithelialization, epidermal cells permanently change shape from pentagonal or hexagonal to irregular forms. This process requires zipper, a gene encoding the Drosophila nonmuscle myosin II heavy chain. Following wounding, myosin II is localized at the wound margin and at the rear end of individual cells located within several rows from the wound hole. The c-Jun N-terminal kinase (JNK) pathway is essential for this myosin II localization. These results suggest that not only the wound leading edge but also the cells lying distal to the leading edge cells actively participate in epithelial cell sheet migration during wound hole closure.

O-GlcNAcylation regulates hyperglycemia-induced GPX1 activation.

Hyperglycemia induces activation of glutathione peroxidase 1 (GPX1), an anti-oxidant enzyme essential for cell survival during oxidative stress. However, the mechanism of GPX1 activation is unclear. Here, we report that hyperglycemia-induced protein glycosylation by O-linked N-acetylglucosamine (O-GlcNAc) is crucial for activation of GPX1 and for its binding to c-Abl and Arg kinases. GPX1 itself is modified with O-GlcNAc on its C-terminus. We also demonstrate that pharmacological injection of the O-GlcNAcase inhibitor NTZ induces GPX1 activation in the mouse liver. Our findings suggest a crucial role for GPX1 and its O-GlcNAc modification in hyperglycemia and diabetes mellitus.

Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria.

Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.

The agrin/perlecan-related protein eyes shut is essential for epithelial lumen formation in the Drosophila retina.

The formation of epithelial lumina is a fundamental process in animal development. Each ommatidium of the Drosophila retina forms an epithelial lumen, the interrhabdomeral space, which has a critical function in vision as it optically isolates individual photoreceptor cells. Ommatidia containing an interrhabdomeral space have evolved from ancestral insect eyes that lack this lumen, as seen, for example, in bees. In a genetic screen, we identified eyes shut (eys) as a gene that is essential for the formation of matrix-filled interrhabdomeral space. Eys is closely related to the proteoglycans agrin and perlecan and secreted by photoreceptor cells into the interrhabdomeral space. The honeybee ortholog of eys is not expressed in photoreceptors, raising the possibility that recruitment of eys expression has made an important contribution to insect eye evolution. Our findings show that the secretion of a proteoglycan into the apical matrix is critical for the formation of epithelial lumina in the fly retina.