Autophagy is a bulk degradation process in eukaryotic cells. Increasing evidence suggest that autophagy has diverse physiological functions, including starvation adaptation, development, immunity, tumor suppression, and cell death. The origin and source of autophagosomal membranes are long-standing questions in the field. Using electron microscopy, we show that the endoplasmic reticulum (ER) associates with early autophagic structures called isolation membranes (IM) in mammalian culture cells. Electron tomography revealed that the ER-associated IM appears as a subdomain of the ER that formed a cradle encircling the IM, and showed that both ER and isolation membranes were interconnected. Our results suggested that autophagosomal membrane originate from ER subdomains, and the ER is a membrane souse for autophagosome formation.
Autophagy is a dynamic cellular process, which can take place in indefinite places within the cytoplasm, making it difficult to predict when and where it occurs. To overcome such difficulties, we have developed a new method to induce autophagy in defined places around polystyrene beads internalized by non-phagocytotic cells, and also developed a method of live cell fluorescence imaging combined with electron microscopy, designated “live CLEM” (correlative light and electron microscopy after live imaging). This technology provides a unique opportunity to visualize dynamic processes of autophagy within the cell in the living state and in high resolution. In this paper, we demonstrate live CLEM imaging of the beads internalized into living cells, and discuss how this method is powerful to understand cellular processes associated with autophagy.
Autophagy-dependent degradation of mitochondria is a fundamental process conserved from yeast to humans. In contrast to starvation-induced, nonselective autophagy responsible for nutrient recycling, selective autophagy, which involves particular cues and receptors, mediates breakdown specific to mitochondria. Although numerous studies highlight that mitochondria-specific autophagy (mitophagy) contributes to mitochondrial homeostasis, the molecular mechanisms underlying this selective clearance process are poorly understood. Our fluorescence and electron microscopy analyses reveal that a substantial fraction of mitochondria are exclusively sequestered, and transported to the vacuole, a lytic compartment, in yeast cells at post-log phase under respiratory conditions. This phenomenon requires Atg11 but not Atg17, proteins acting as scaffolds crucial for selective and nonselective autophagy pathways, respectively. Using a genome-wide visual screen, we identified Atg32, a protein essential for mitophagy in yeast. During respiratory growth, Atg32 is highly expressed, likely in response to oxidative stress, and anchored to the surface of mitochondria. We also demonstrate that Atg32 interacts with Atg8 and Atg11, proteins critical for recognition of cargo receptors. Notably, Atg32 contains WXXI/L/V, a conserved motif that serves as a binding site for the Atg8 family members. We propose that Atg32 is a transmembrane receptor that directs the autophagy machinery to mitochondria.
The autophagy research is expanding rapidly and the relationship between autophagy and lipid droplets is attracting much attention. However, whether the lipid droplet is just one of many organelles that are processed by autophagy or an essential structure for autophagic induction has not been resolved. In this article, we introduced and commented on several recent papers dealing with lipid droplets and autophagy.
During oogenesis, oocytes accumulate maternal mRNAs and proteins. After fertilization however, these maternal products are rapidly degraded and new products encoded by the zygotic genome are synthesized. This transition is critical for preimplantation embryonic development. Since the transition occurs rapidly after fertilization, it is reasonable that large scale of degradation systems could be involved in this transition. Autophagy is a cytoplasmic degradation system mediated by lysosome. Whether autophagy is essential for early embryogenesis has been controversial. We recently found that autophagy is highly induced soon after fertilization. Using oocyte-specific Atg5 knockout mice, we revealed that autophagy-deficiency caused embryonic lethality at four-to-eight cell stage embryos. Thus, our results suggested that autophagy is essential for oocyte-to-embryo transition.
Atg5 and Atg7 are considered as essential molecules for induction of macroautophagy. However, we found that cells lacking Atg5 or Atg7 can still form autophagosomes/autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stresses. Although lipidation of LC3 is accepted to be a good indicator of macroautophagy, it did not occur during the Atg5/Atg7-independent alternative macroautophagy. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of the phagophore with vesicles derived from the trans-Golgi and late endosomes. Mammalian macroautophagy can occur via at least two different pathways, which are an Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.
Mammalian muscle spindles are generally composed of two types of intrafusal muscle fibers: nuclear bag fibers and nuclear chain fibers. Each intrafusal muscle fiber is innervated by sensory and motor nerves. However, the distribution of nerve endings is different between nuclear bag fibers and nuclear chain fibers. The nuclear bag fibers are supplied by sensory (primary) endings in the equatorial region and motor endings from the juxtaequatorial region to the polar region. On the other hand, the nuclear chain fibers are widely innervated by sensory (primary and secondary) endings from the equatorial region to the polar region and motor endings are restricted only in the polar region. Sensory endings generally lie on the surface of the muscle fibers in shallow grooves. However, in the intrinsic laryngeal muscle such as human being, guinea pig and marmoset, sensory endings branch and deeply penetrate into the muscle fibers. Motor endings are classified into at least three types. In addition to sensory endings and motor endings, multiaxonal endings consisting of a bundle of axons occasionally exist in the muscle spindles of the adult Chinese hamster. From a structural organization similar to the autonomic nerves, these multiaxonal endings are considered to be autonomic in nature.
In the present paper, recent progress of a novel method called electron tomography in the visualisation of dislocations was reviewed. The method of obtaining three-dimensional structure of dislocations was explained first. The applications of the electron tomography combining with electron microscopy such as dark-field, STEM, HVEM and dual-axis were also demonstrated.
Electron cryo-tomography single particle analysis is a method to distinguish heterogeneous bio-macromolecules and analyze them, which take various conformational changes in the sample solution. We apply this method for studying the structural changes of the T7-like virus, P-SSP7 during the infection of marine cyanobacteria, Prochrolococcus MED4. We found that there are three states in the viral adsorption to the cell surface. The virus changes the conformation of the spike fibers with these steps and finally injects the viral DNA into the host cell through the tail vertically connected to the cell surface. In addition, a comparison with the high-resolution structures from the conventional single particle analysis showed a relationship between the conformational changes of the spike fibers and the DNA gating in the tail. Here, we describe the methods of image processing and present the biological results.
Electron optical systems for aberration correction in electron microscopy are outlined starting from Scherzer theorem established in 1936. There are two types of third-order spherical aberration correction-lens systems that used multipoles. One is the so-called hexapole corrector developed by Rose, Haider et al., and the other is the quadrupole−octupole corrector. Self-aligned quadrupole correction-lens is introduced to simplify the construction of the quadrupole−octupole corrector. Axial chromatic aberration can be also corrected by combination of electrostatic and magnetic quadrupoles, or electrostatic multipole system satisfying optimum relation between the axial potential and the quadrupole strengths.
Multi-biprism electron interferometry which utilizes two or three electron biprisms in the electron optical system simultaneously, is introduced as being based on conventional electron interferometry using a single electron biprism in its optical system. Electron biprisms in the newly developed interferometry, are installed into real space and reciprocal space, separately and simultaneously in order to control two electron waves independently in their relative propagate angle and their overlap position in the interference optical system. Several important parameters of interferogram, such as fringe spacing, interference width and orientation of interference fringes which is newly introduced by the interferometer, are able to control independently and flexibly. The construction of the interference optical systems and their operation mechanism are discussed in this paper.
The goal of specimen preparation for morphological investigation is to preserve the tissue or cell in the native state with which we can correlate structure and function. Cryofixation is generally accepted as the best initial fixation step to preserve not only the fine structure but also the antigenicity in biological samples. At the ultrastructural level, high-pressure freezing technique is currently the most reliable method to obtain a deep vitreous freezing that enable us to study complex biological samples with improved ultrastructural preservation. Here we introduce the application of high-pressure freezing/freeze substitution onto ultrastructural study of gastric mucosa which provides excellent ultrastructures and antigenicities of not only the epithelial cells of rat gastric glands but also the intraluminal exocytosed contents.
Present situation of developing low-energy SEM mounted with a single-atom electron source has been reviewed. In the electron gun, adjustment of direction alignment of an electron beam is possible in addition to the adjustment of beam position. The highly efficient electron gun was realized; SEM observation was realized at several nA of the total emission current, which is three orders of magnitude as small as those of conventional FE guns. Improvement in the spatial resolution is in progress.
We imeplemented the method of time-delayed feedback control to a commercial dynamic-mode atomic force microscope for suppressing chaotic oscillation caused by the nonlinear tip-sample interaction force. Cantilever oscillation was successfully stabilized by applying our prototype time-delayed feedback controller with the magnetic cantilever excitation method.