Atsushi Matsuda

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.

Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones.

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

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.

The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

ABSTRACT Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena ΔDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.

Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast

It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or ‘knobs. Knob regions span ∼50u2009kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins

Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.

Dual-axis target mapping and automated sequential acquisition of dual-axis EM tomographic data.

Dual-axis electron microscopic tomography minimizes the missing wedge-induced resolution loss by taking two complementary tilt data sets of the same target along two orthogonal axes. The potential of this powerful approach has been hampered by the practical challenges inherent in finding the original targets that are dramatically displaced due to non-eucentric specimen rotation. Not only is the manual search for the original targets time consuming and tedious but the added dose during manual searching is uncontrollable. We have developed a hierarchical alignment scheme that allows tomographic data to be collected from an arbitrary number of target sites in one grid orientation and then to find and collect orthogonal data sets with little or no user intervention. Inspired by the successful multi-scale mapping in Leginon, our alignment is performed in three levels to gradually pinpoint the original targets. At the lowest level the grid lattice is used to determine the rotation angle and translational shift resulting from specimen rotation via auto- and cross-correlative analysis of a pair of atlas maps constructed before and after specimen rotation. The target locations are further refined at the next level using a pair of smaller atlas maps. The final refinement of target positions is done by aligning the target contained image tiles. Given the batch processing nature of this hierarchical alignment, multiple targets are initially selected in a group and then sequentially acquired. Upon completion of the data collection on all the targets along the first axis and after specimen rotation, the hierarchical alignment is performed to relocate the original targets. The data collection is then resumed on these targets for the second axis. Therefore, only one specimen rotation is needed for collecting multiple dual-axis tomographic data sets. The experiment of acquiring 20S Proteasomes dual-axis tomographic data sets in vitreous ice at 86,000x CCD magnification on our FEI Tecnai Polara TF30 electron microscope has suggested that the developed scheme is very robust. The extra doses for finding and centering the original targets are almost negligible. This scheme has been integrated into UCSF Tomography software suite that can be downloaded at www.msg.ucsf.edu/tomography free for academic use.

The SUMO Pathway Is Developmentally Regulated and Required for Programmed DNA Elimination in Paramecium tetraurelia

ABSTRACT Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.