Hiroko Osakada

Artificial induction of autophagy around polystyrene beads in nonphagocytic cells

Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to ∼80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.

Inner nuclear membrane protein Ima1 is dispensable for intranuclear positioning of centromeres.

Inner nuclear membrane (INM) proteins play a role in spatial organization of chromosomes within the nucleus. In the fission yeast Schizosaccharomyces pombe, Sad1, an INM protein of the conserved SUN‐domain family, plays an active role in moving chromosomes along the nuclear membranes during meiotic prophase. Ima1 is another conserved INM protein recently identified. A previous study claimed that Ima1 is essential for mitotic cell growth, linking centromeric heterochromatin to the spindle‐pole body. However, we obtained results contradictory to the previously proposed role for Ima1: Ima1 was dispensable for mitotic cell growth or centromere positioning. This discrepancy was attributed to incorrect ima1 deletion mutants used in the previous study. Our results show that Ima1 collaborates with two other conserved INM proteins of the LEM‐domain family that are homologous to human Man1 and Lem2. Loss of any one of three INM proteins has no effect on mitotic cell growth; however, loss of all these proteins causes severe defects in mitotic cell growth and nuclear membrane morphology. Considering that all three INM proteins interact with Sad1, these results suggest that Ima1, Lem2 and Man1 play at least partially redundant roles for nuclear membrane organization.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live‐cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior‐posterior polarized nuclei: MAC‐specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC‐specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light‐electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC‐specific NPCs in the division‐failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

Significance Rapid detection of invasion of exogenous materials and subsequent responses are important for living organisms to survive hazards, such as pathogen infection. Understanding cellular responses against exogenous DNA provides clues not only for controlling pathogen infections that bring exogenous DNA into host cells, but also for designing efficient DNA delivery vectors for transgene expression. Here, by monitoring the invasion of exogenous DNA-coated polystyrene beads into living cells, we show that barrier-to-autointegration factor detects exogenous DNA immediately after its appearance at endosome breakdown and plays a role in DNA avoiding autophagy. These findings provide new insights into the mechanisms by which a cell detects and responds to exogenous double-stranded DNA. Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell’s response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF+ DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in the ciliate Tetrahymena

ABSTRACT The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins and subcellular localization analysis of GFP-fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107–Nup160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either the MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on the compositional and structural specificity of NPCs. Summary: There are compositional and structural differences in the nuclear pore complexes present in the functionally differentiated macronucleus and micronucleus within the single cytoplasm of ciliated protozoa.

Monoclonal Antibodies Recognize Gly-Leu-Phe-Gly Repeat of Nucleoporin Nup98 of Tetrahymena, Yeasts, and Humans

Nucleoporin Nup98, an essential component of the nuclear pore complex, has multifunctional roles in nuclear functions including transcriptional regulation and nucleocytoplasmic transport. These functions mostly depend on a Gly-Leu-Phe-Gly (GLFG) sequence appearing repetitively in the N-terminal region of Nup98. As the GLFG sequence is well conserved among Nup98s from a wide variety of species including humans, yeasts, and ciliates such as Tetrahymena thermophila, a specific antibody that recognizes the GLFG sequence is expected to detect various Nup98s from a wide-range of species. To generate monoclonal antibodies specific to the GLFG repeat of Nup98, we used two synthetic polypeptides derived from the macronuclear Nup98 of T. thermophila as an antigen. We obtained two monoclonal antibodies (MAbs), 13C2 and 21A10, that recognize Nup98s in indirect immunofluorescence staining and Western blot analysis of T. thermophila. Peptide array analysis of these monoclonal antibodies located the position of their epitopes at or near GLFG residues: the epitope recognized by the 13C2 MAb is FGxxN (x being any amino acid), and the epitope recognized by the 21A10 MAb is GLF. As expected by their epitopes, these monoclonal antibodies also recognize Nup98 homologs expressed by human cells and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, indicating that 13C2 and 21A10 MAbs recognize Nup98 epitopes common to phylogenetically distinct organisms. Thus, these MAbs are useful in studying a wide variety of biological phenomena that involve Nup98, ranging from ciliate nuclear dimorphism to NUP98-related human leukemia.

Early entry and deformation of macropinosomes correlates with high efficiency of decaarginine-polyethylene glycol-lipid-mediated gene delivery.

Decaarginine‐polyethylene glycol‐conjugated 3,5‐bis(dodecyloxy)benzamide/plasmid DNA [Arg10‐polyethylene glycol (PEG)‐lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated.

Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment

Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

Lysosome-related organelles (LROs) are secretory organelles formed by convergence between secretory and endosomal trafficking pathways. In Tetrahymena, secretory vesicles that resemble dense core granules are a new class of LROs whose synthesis depends on a conserved syntaxin required for heterotypic fusion and AP-3 for maturation.