Tokuko Haraguchi

Autophagosomes form at ER–mitochondria contact sites

Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate—the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER–mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER–mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER–mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER–mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER–mitochondria contact site is important in autophagosome formation.

Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells

Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non‐homologous end‐joining (NHEJ) DNA double‐strand break (DSB) repair pathways. Vertebrate Mre11 is essential. Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification. Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies. Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair. Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11‐independent component of metazoan NHEJ.

Dynamics of Homologous Chromosome Pairing during Meiotic Prophase in Fission Yeast

Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis. It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast. Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast. Homologous loci exhibited a dynamic process of association and dissociation during the time course of meiotic prophase. Lack of nuclear oscillation reduced association frequency for both centromeric and arm regions of the chromosome. Lack of telomere clustering or recombination reduced association frequency at arm regions, but not significantly at centromeric regions. Our results indicate that homologous chromosomes are spatially aligned by oscillation of telomere-bundled chromosomes and physically linked by recombination at chromosome arm regions; this recombination is not required for association of homologous centromeres.

Molecular Communication by Tadashi Nakano

This paper describes research challenges in Molecular Communication, a new and interdisciplinary research area that spans the nanotechnology, biotechnology, and communication technology. Molecular communication allows nanomachines to communicate using molecules as a communication carrier. Key research challenges include controlled propagation of carrier molecules, encoding/ decoding of information onto information molecules, and transmission/reception systems for carrier/information molecules. The authors of this paper are currently investigating the feasibility of molecular communication.

Identification and Characterization of Nuclear Pore Complex Components in Arabidopsis thaliana

Interactive proteomics technology was used to identify Arabidopsis nucleoporins, which are components of the nuclear pore complex (NPC). Nucleoporin domain organization is similar in plants, vertebrates, and yeast. This finding suggests that most NPC structures are conserved throughout eukaryotes. The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a crucial process for various cellular activities. The NPC comprises ~30 nucleoporins and is well characterized in vertebrates and yeast. However, only eight plant nucleoporins have been identified, and little information is available about the complete molecular structure of plant NPCs. In this study, an interactive proteomic approach was used to identify Arabidopsis thaliana nucleoporins. A series of five cycles of interactive proteomic analysis was performed using green fluorescent protein (GFP)-tagged nucleoporins. The identified nucleoporins were then cloned and subcellular localization analyses were performed. We found that the plant NPC contains at least 30 nucleoporins, 22 of which had not been previously annotated. Surprisingly, plant nucleoporins shared a similar domain organization to their vertebrate (human) and yeast (Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins exhibited higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358], Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis failed to identify a vertebrate homolog for this protein. Interestingly, Nup136-GFP showed greater mobility on the nuclear envelope than did other nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant development. These findings provide valuable new information about plant NPC structure and function.

Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe

In fission yeast meiotic prophase, telomeres are clustered near the spindle pole body (SPB; a centrosome‐equivalent structure in fungi) and take the leading position in chromosome movement, while centromeres are separated from the SPB. This telomere position contrasts with mitotic nuclear organization, in which centromeres remain clustered near the SPB and lead chromosome movement. Thus, nuclear reorganization switching the position of centromeres and telomeres must take place upon entering meiosis. In this report, we analyze the nuclear location of centromeres and telomeres in genetically well‐characterized meiotic mutant strains. An intermediate structure for telomere‐centromere switching was observed in haploid cells induced to undergo meiosis by synthetic mating pheromone; fluorescence in situ hybridization revealed that in these cells, both telomeres and centromeres were clustered near the SPB. Further analyses in a series of mutants showed that telomere‐centromere switching takes place in two steps; first, association of telomeres with the SPB and, second, dissociation of centromeres from the SPB. The first step can take place in the haploid state in response to mating pheromone, but the second step does not take place in haploid cells and probably depends on conjugation‐related events. In addition, a linear minichromosome was also co‐localized with authentic telomeres instead of centromeres, suggesting that telomere clustering plays a role in organizing chromosomes within a meiotic prophase nucleus.

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells

CENP‐H has recently been discovered as a constitutive component of the centromere that co‐localizes with CENP‐A and CENP‐C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP‐H in centromere function and assembly by generating a conditional loss‐of‐function mutant in the chicken DT40 cell line. In the absence of CENP‐H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP‐H‐deficient cells demonstrated that CENP‐H is necessary for CENP‐C, but not CENP‐A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere‐specific histone CENP‐A is an early event that occurs independently of CENP‐C and CENP‐H.

Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells.

Nuf2 and Hec1 are evolutionarily conserved centromere proteins. To clarify the functions of these proteins in vertebrate cells, we characterized them in chicken DT40 cells. We generated GFP fusion constructs of Nuf2 and Hec1 to examine in detail the localization of these proteins during the cell cycle. We found that Nuf2 is associated with Hec1 throughout the cell cycle and that this complex is localized to the centrosomes during G1 and S phases and then moves through the nuclear membrane to the centromere in G2 phase. During mitosis, this complex is localized to the centromere. We also created conditional loss-of-function mutants of Nuf2 and Hec1. In both mutants, the cell cycle arrested at prometaphase, suggesting that the Nuf2-Hec1 complex is essential for mitotic progression. The inner centromere proteins CENP-A, -C, and -H and checkpoint protein BubR1 were localized to chromosomes in the mutant cells arrested at prometaphase, but Mad2 localization was abolished. Furthermore, photobleaching experiments revealed that the Nuf2-Hec1 complex is stably associated with the centromere and that interaction of this complex with the centrosome is dynamic.

Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library

Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)‐fusion genomic DNA library of S. pombe.

Cellular stresses induce the nuclear accumulation of importin α and cause a conventional nuclear import block

We report here that importin α accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin α appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin α. In addition, nuclear retention and the importin β/Ran-independent import of importin α also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin α from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin α is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin α can be involved in a common physiological response to various stress conditions.