Publisher Summary Advances in the understanding of the formation, structure, and chemistry of the mammalian kinetochore and its associated fiber are summarized in the chapter. The initiation of chromosome movement during cell division can be correlated with the formation of a fiber, composed primarily of microtubules (MTs) and associated proteins that connect each chromosome to the polar area of the spindle. The region on the chromosome where the MTs attach is the kinetochore, and the MTs themselves are known as “kinetochore MTs” (K-MTs). The origin of K-MTs is consistent with the morphological changes that occur within the astral spindle during prometaphase, with the structure of the K-fiber, with the ultrastructural data on prometaphase in many types of cells, with the in vivo nucleation data, and with the in vivo polarity determinations. The origin of K-MTs offers an explanation for various in vivo observations that have yet to be explained by a mechanism of K-MT formation, based solely on the nucleation of MTs by the kinetochore.

The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber.

Publisher Summary Mitosis is defined as all those types of nuclear division that produce two, or rarely more, daughter nuclei, each containing a chromosome complement approximately similar to that of the original nucleus. The greatest range of variations by which mitosis is accomplished, occurs in the protistan and fungal kingdoms, some members of which are probably most similar to the ancestors of higher plants and animals. The variations in the higher organisms are secondarily derived from the division patterns of typical plants and animals. This chapter discusses the characteristics and evolution of mitosis. The efficiency of mitosis consists of two basic components; the frequency with which each daughter nucleus receives the necessary complete genome complement (genetic efficiency) and the amount of energy and materials expended in the synthesis and operation of the mitotic apparatus. The chapter also discusses the use of mitosis as a phylogenetic marker. It is applicable to all eukaryotic cells and thus is valuable across boundaries where other structures are absent on one side and present in various forms on the other side.

Variant mitoses in lower eukaryotes: indicators of the evolution of mitosis.

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.

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Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation.

The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/febs.13307

DNA structure and function

When metaphase PtK1 cells are cooled to 6–8 ° C for 4–6 h the free, polar, and astral spindle microtubules (MTs) disassemble while the MTs of each kinetochore fiber cluster together and persist as bundles of cold-stable MTs. These cold-stable kinetochore fibers are similar to untreated kinetochore fibers in both their length (i.e., 5–6 μm) and in the number of kinetochore-associated MTs (i.e., 20–45) of which they are comprised. Quantitative information concerning the lengths of MTs within these fibers was obtained by tracking individual MTs between serial transverse sections. Approximately 1/2 of the kinetochore MTs in each fiber were found to run uninterrupted into the polar region of the spindle. It can be inferred from this and other data that a substantial number of MTs run uninterrupted between the kinetochore and the polar region in untreated metaphase PtK1 cells.

The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells.

Fixation and staining of F-actin and microfilaments using tannic acid

Birefringence and fine structure of spindles in spermatocytes of Nephrotoma suturalis at metaphase of first meiotic division

Analysis of birefringence and ultrastructure of spindles in primary spermatocytes of Nephrotoma suturalis during anaphase

The micronuclear mitotic spindle of Tetrahymena pyriformis contains several distinctive elements. The 150 or so continuous microtubules (MTs) from a peripheral sheath just inside the inner nuclear membrane while the kinetochore bundles traverse the center of the nucleoplasm. A new set of microtubules, the separation spindle, appears during the 10-fold nuclear elongation which occurs during late anaphase. Both the separation spindle and peripheral sheath MTs are present in the macronucleus during macronuclear division but there are no definite kinetochore MTs. In the crescent stage of meiotic prophase both peripheral sheath and kinetochire MTs are present in the micronucleus. By using our own, and other workers', data in conjunction with the phylogenetic scheme for the ciliates which has been designed by Corliss (1974, 1975), we have attempted to trace out the evolutionary history of the various elements of the ciliate mitotic spindle. For example, the micronuclear separation spindle can be followed, from its point of origin within the primitive gymnostomes, throughout the phylum. The separation spindle of the macronucleus, by contrast, is lost at the level of the heterotrichs. Similarly, the anaphase breakdown and reconstitution of the nuclear envelope, which occurs in the primitive Loxodes magnus , can be followed up some phyletic branches, such as the heterotrichs, but in the suctorians and some other groups this feature seems to have been lost. Tracing the evolution of the ciliate mitotic spindle is made very difficult both by the incompleteness of the data and by what appear to be a number of cases of secondary reduction and parallel evolution. In general, however, the evolution of mitosis correlates well with the phylogeny of the ciliates constructed by Corliss and we consider this an independent substantiation of the general correctness of his phylogeny.

Mitosis and early meiosis in Tetrahymena pyriformis and the evolution of mitosis in the phylum ciliophora

An investigation of the spindle apparatus of crane-fly (Nephrotoma suturalis) spermatocytes has been undertaken using methods that permit combined light and electron microscopy of selected cells. At the ultrastructural level, spindles contain microtubules in a granular matrix. Microtubules have been classified as kinetochore microtubules (which connect to kinetochores of chromosomes) and non-kinetochore microtubules (not attached to kinetochores). Kinetochore microtubules are distributed in densely packed bundles, which are the birefringent chromosomal fibers seen in living cells. Actin filaments were not observed in spindles of unglycerinated cells or in cells fixed in glutaraldehyde containing tannic acid, which negatively stains F-actin in situ and thus can be used to aid the localization of actin filaments in non-muscle cells. The absence of actin filaments in the spindle coupled with their presence in the "contractile ring" of spermatocytes fixed during cytokinesis is evidence against the hypothesis that chromosome movements are microfilament-based. The results are compatible with the hypothesis that microtubules are involved in the mechanism of chromosome transport. The details of that mechanism remain to be clarified.

James R. LaFountain

Papers

Fixation and staining of F-actin and microfilaments using tannic acid

Birefringence and fine structure of spindles in spermatocytes of Nephrotoma suturalis at metaphase of first meiotic division

Analysis of birefringence and ultrastructure of spindles in primary spermatocytes of Nephrotoma suturalis during anaphase

Mitosis and early meiosis in Tetrahymena pyriformis and the evolution of mitosis in the phylum ciliophora

What moves chromosomes, microtubules or microfilaments?