Cryo-EM structure of a molluscan hemocyanin suggests its allosteric mechanism.

Hemocyanins are responsible for transporting O2 in the arthropod and molluscan hemolymph. Haliotis diversicolor molluscan hemocyanin isoform 1 (HdH1) is an 8 MDa oligomer. Each subunit is made up of eight functional units (FUs).

Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle.

The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction.

Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus.

Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic photosynthetic microorganism. Single-particle cryo-electron microscopy yields icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6-A and 9-A resolution, respectively.

Electron microscopy, immunostaining, cytoskeleton visualization, in situ hybridization, and three-dimensional reconstruction of Xenopus oocytes.

Although the overwhelming development of molecular techniques in recent decades has made ultrastructural studies less popular, to the point that ultrastructural interpretation is becoming a dying art, it still remains an indispensable tool for cell and developmental biologists. The introduction of EM-immunocytochemistry and three-dimensional visualization methods allows us to complement the knowledge gained from ultrastructural and molecular approaches. Because the first clues about the functions of newly discovered genes often come from the subcellular localization patterns of their proteins or RNAs, in this chapter we describe the methods that allow for precise ultrastructural localization and visualization of protein and RNA molecules within the compartments, organelles, and cytoskeleton of Xenopus oocytes.

Organization of cytokeratin cytoskeleton and germ plasm in the vegetal cortex of Xenopus laevis oocytes depends on coding and non-coding RNAs: three-dimensional and ultrastructural analysis.

Recent studies discovered a novel structural role of RNA in maintaining the integrity of the mitotic spindle and cellular cytoskeleton. In Xenopus laevis, non-coding Xlsirts and coding VegT RNAs play a structural role in anchoring localized RNAs, maintaining the organization of the cytokeratin cytoskeleton and germinal granules in the oocyte vegetal cortex and in subsequent development of the germline in the embryo. We studied the ultrastructural effects of antisense oligonucleotide driven ablation of Xlsirts and VegT RNAs on the organization of the cytokeratin, germ plasm and other components of the vegetal cortex.

Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud.

During Xenopus oogenesis, the message transport organizer (METRO) pathway delivers germinal granules and localized RNAs to the vegetal cortex of the oocyte via the mitochondrial cloud (Balbiani body). According to the traditional model, the mitochondrial cloud is thought to break up at the onset of vitellogenesis and the germinal granules and METRO-localized RNAs are transported within the mitochondrial cloud fragments to the vegetal cortex of the oocyte. We used light and electron microscopy in situ hybridization and three-dimensional reconstruction to show that germinal granules and METRO-localized RNAs are delivered to the oocyte cortex before the onset of mitochondrial cloud fragmentation and that the delivery involves accumulation of localized RNAs and aggregation of germinal granules at the vegetal tip of the mitochondrial cloud and subsequent internal expansion of the mitochondrial cloud between its animal (nuclear) and vegetal tips, which drives the germinal granules and METRO-localized RNAs toward the vegetal cortex.

Formation, architecture and polarity of female germline cyst in Xenopus.

Little is known about the formation of germline cyst and the differentiation of oocyte within the cyst in vertebrates. In the majority of invertebrates in the initial stages of gametogenesis, male and female germ cells develop in full synchrony as a syncytia of interconnected cells called germline cysts (clusters, nests). Using electron microscopy, immunostaining and three-dimensional reconstruction, we were able to elucidate the process of cyst formation in the developing ovary of the vertebrate Xenopus laevis.

Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA.