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.

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.

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.