Necrosensor: a genetically encoded fluorescent sensor for visualizing necrosis in Drosophila.

Historically, necrosis has been considered a passive process, which is induced by extreme stress or damage. However, recent findings of necroptosis, a programmed form of necrosis, shed a new light on necrosis. It has been challenging to detect necrosis reliably in vivo, partly due to the lack of genetically encoded sensors to detect necrosis. This is in stark contrast with the availability of many genetically encoded biosensors for apoptosis. Here we developed Necrosensor, a genetically encoded fluorescent sensor that detects necrosis in Drosophila, by utilizing HMGB1, which is released from the nucleus as a damage-associated molecular pattern (DAMP). We demonstrate that Necrosensor is able to detect necrosis induced by various stresses in multiple tissues in both live and fixed conditions. Necrosensor also detects physiological necrosis that occurs during spermatogenesis in the testis. Using Necrosensor, we discovered previously unidentified, physiological necrosis of hemocyte progenitors in the hematopoietic lymph gland of developing larvae. This work provides a new transgenic system that enables in vivo detection of necrosis in real time without any intervention.

Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Epigenetic plasticity safeguards heterochromatin configuration in mammals.

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating.

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.

Fis1 acts as a mitochondrial recruitment factor for TBC1D15 that is involved in regulation of mitochondrial morphology.

In yeast, C-tail-anchored mitochondrial outer membrane protein Fis1 recruits the mitochondrial-fission-regulating GTPase Dnm1 to mitochondrial fission sites. However, the function of its mammalian homologue remains enigmatic because it has been reported to be dispensable for the mitochondrial recruitment of Drp1, a mammalian homologue of Dnm1. We identified TBC1D15 as a Fis1-binding protein in HeLa cell extracts. Immunoprecipitation revealed that Fis1 efficiently interacts with TBC1D15 but not with Drp1. Bacterially expressed Fis1 and TBC1D15 formed a direct and stable complex. Exogenously expressed TBC1D15 localized mainly in cytoplasm in HeLa cells, but when coexpressed with Fis1 it localized to mitochondria. Knockdown of TBC1D15 induced highly developed mitochondrial network structures similar to the effect of Fis1 knockdown, suggesting that the TBC1D15 and Fis1 are associated with the regulation of mitochondrial morphology independently of Drp1. These data suggest that Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells.

Mitochondrial fusion and inheritance of the mitochondrial genome.

Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi,including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents.Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade,a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum.A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism.Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum.An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion,mediated by mF. Physarum is a unique organism for studying mitochondrial fusion.