Transmission electron microscopy method providing high resolution in the cell section without radiation damage.

Several new features of mitochondrial nucleoid and its surroundings in mammalian cells were described previously (Prachar, 2016). Very small details were observed using the improved transmission electron microscopy method, as described in the article. In the meantime, the method has again been improved to 2 Å resolutions in the cell section. The method described in detail in the present work is documented on the same records that were published in lower resolution in the work Prachar (2016), enabling comparison of the achieved resolution with the previous one. New records are also presented, showing extremely high resolution and thus implying the importance of the method. Potential use of this method in different fields is suggested.

Ultrastructure of mitochondrial nucleoid and its surroundings.

Mitochondrial nucleoids (hereafter nucleoids) contain genetic information, mitochondrial DNA, prerequisite for mitochondrial functioning, particularly information required for mitochondrial electron transport. To understand nucleoid functioning, it is imperative to know its ultrastructure and dynamics in the context of the actual mitochondrial state. In this study, we document the internal structure, different positions of nucleoids inside the mitochondrial tube and their different morphology. The nucleoid cores appear in section as circular or slightly oval objects ranging from 50 to 100 nm in diameter. They are mainly located in the matrix between cristae inside the mitochondrial tube but they are also frequently found close to the inner mitochondrial surface. In tightly packed form, their interior exhibits sophisticated nucleoprotein regularity. The core surroundings form an electron-lucent thick layer which is probably partitioned into separate chambers. We suggest that the morphology of nucleoids mirrors the mode of energy production, glycolysis versus oxidative phosphorylation. The new high resolution transmission electron microscopy method enabled us to obtain morphological characteristics on yet unpublished level.

Spoke ring and anchorage of nuclear pore complex revealed by high resolution transmission electron microscopy.

Using several different methods, the nuclear pore complex (NPC) was shown to be anchored in the nuclear envelope into the specific curved region, called pore membrane. Three transmembrane nucleoporins in the equatorial region of NPC contain hydrophobic stretches, which exhibit the ability to intersect the phospholipid bilayer. Using transmission electron microscopy, we observed three different evaluable morphological situations in the section through the NPC spoke ring (SR). We suppose that some sections are directed through one type of subunit that is responsible for anchoring. Other sections are directed through the second type of subunit that may provide pore membrane bending. Finally, the spoke ring is sectioned between aforementioned subunits where the pore membrane is best preserved. The proposed anchor is represented by the chains of protein complexes which replace phospholipid bilayer in a relatively large area. Second subunit, presumed bending module is represented by the bundles of chains copying the shape of the pore membrane from the side of the NPC. This work is based on very high resolution resulting in unique and complicated images of tangled and cut off protein chains, nevertheless, it provides insight into how some proteins interact with or replace the membrane.

Structural features of transversal barrier in central channel of nuclear pore complex.

The most important entity of the selective behavior of the nuclear pore complex (NPC) is considered to be the matter called "barrier," "meshwork" or "sieve." This part of NPC has not been well elucidated by using electron microscopy methods to date. In the present study, we demonstrated the presence of a coherent transversal barrier in the central channel of NPC, using high resolution transmission electron microscopy. It was found that the barrier is located in the middle of the central channel, i.e. at the level where the outer and inner nuclear membranes fuse. The thickness of this layer is evidently different in various NPCs and usually varies between 20 and 30 nm and its diameter is approximately 40 nm. The cytoplasmic and nuclear surfaces of the barrier are roughly parallel and plane. Moreover we suggest that the barrier may not be interrupted by any channel(s), at least not with a diameter above 10 nm. Further various appearances of the central channel with different particles were observed, presumably cargos and karyopherins captured in transit. A different type of central channel barrier with lipid bilayer membrane-like appearance is also discussed.

Mouse and human mitochondrial nucleoid--detailed structure in relation to function.

The independent mitochondrial genetic information is organized in so-called mitochondrial nucleoids that, in vertebrates, typically contain 5-7 genetic units. The total number of nucleoids per cell is several hundred in cultured cells. Mitochondrial nucleoids, similarly to the whole mitochondrial network, have recently been successfully and extensively visualized using fluorescent and confocal microscopy. In the present work, we show high-resolution micrographs of mouse and human mitochondrial nucleoids obtained by transmission electron microscopy. Position in the mitochondria, size, general appearance and other properties of the human nucleoids appear the same as those of mouse nucleoids, and all observations are also in full agreement with the results obtained in different laboratories using different approaches. Most of nucleoids are located inside mitochondrial tubes. However, we show directly that certain part of the nucleoids close to inner membrane is bound to the complex of molecules that crosscut both, the inner and the outer mitochondrial membranes. Nucleoids in cells starving for serum are mostly more dense than those in dividing cells. We discuss the position, appearance and other properties of the nucleoids in relation to functional stage. Other electron-dense structures inside mitochondria that could be erroneously considered to be mitochondrial nucleoids are also described.