During epithelial cell proliferation, planar alignment of the mitotic spindle coordinates the local process of symmetric cell cleavage with the global maintenance of polarized tissue architecture. Although the disruption of planar spindle alignment is proposed to cause epithelial to mesenchymal transition and cancer, the in vivo mechanisms regulating mitotic spindle orientation remain elusive. Here we demonstrate that the actomyosin cortex and the junction-localized neoplastic tumour suppressors Scribbled and Discs large 1 have essential roles in planar spindle alignment and thus the control of epithelial integrity in the Drosophila imaginal disc. We show that defective alignment of the mitotic spindle correlates with cell delamination and apoptotic death, and that blocking the death of misaligned cells is sufficient to drive the formation of basally localized tumour-like masses. These findings indicate a key role for junction-mediated spindle alignment in the maintenance of epithelial integrity, and also reveal a previously unknown cell-death-mediated tumour-suppressor function inherent in the polarized architecture of epithelia.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae. Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

No abstract

Caspases are at the core of executing apoptosis by orchestrating cellular destruction with proteolytic cascades. Caspase-mediated proteolysis also controls diverse nonlethal cellular activities such as proliferation, differentiation, cell fate decision, and cytoskeletal reorganization. During the last decade or so, genetic studies of Drosophila have contributed to our understanding of the in vivo mechanism of the non-apoptotic cellular responses in developmental contexts. Furthermore, recent studies using C. elegans suggest that apoptotic signaling may play unexpected roles, which influence ageing and normal development at the organism level. In this review, we describe how the caspase activity is elaborately controlled during vital cellular processes at the level of subcellular localization, the duration and timing to avoid full apoptotic consequences, and also discuss the novel roles of non-apoptotic caspase signaling in adult homeostasis and physiology.

In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H, Na, and Cl. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl or SO. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl and SO with a subsequent secondary inward H movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl and from 542 to 556 nm for SO, and the affinities of Cl and SO were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO) transporter and report its spectroscopic characteristics.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29 (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pK values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.

The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.

Tissue remodeling involves collective cell movement, and cell proliferation and apoptosis are observed in both development and disease. Apoptosis and proliferation are considered to be closely correlated, but little is known about their coordinated regulation in physiological tissue remodeling in vivo. The replacement of larval abdominal epidermis with adult epithelium in Drosophila pupae is a simple model of tissue remodeling. During this process, larval epidermal cells (LECs) undergo apoptosis and are replaced by histoblasts, which are adult precursor cells. By analyzing caspase activation at the single-cell level in living pupae, we found that caspase activation in LECs is induced at the LEC/histoblast boundary, which expands as the LECs die. Manipulating histoblast proliferation at the LEC/histoblast boundary, either genetically or by UV illumination, indicated that local interactions with proliferating histoblasts triggered caspase activation in the boundary LECs. Finally, by monitoring the spatiotemporal dynamics of the S/G 2 /M phase in histoblasts in vivo, we found that the transition from S/G 2 phases is necessary to induce nonautonomous LEC apoptosis at the LEC/histoblast boundary. The replacement boundary, formed as caspase activation is regulated locally by cell-cell communication, may drive the dynamic orchestration of cell replacement during tissue remodeling.