Chromosome attachments

The foregoing description overlooks the extreme complexity of mitosis, each stage of which must occur with precision and in the correct sequence 222-2253 The replication of DNA, which takes place in the S phase of the cell cycle (and is discussed in Chapter 27) must be completed before mitosis begins. This is followed by condensation of the DNA into chromosomes (Chapter 27), breakdown of the nuclear membrane,226 2263 assembly of the kinetochores by which the chromosomes attach to the spindle,222 assembly of the spindle, attachment of chromosomes to the spindle, segregation of the chromosomes to opposite poles in anaphase, and finally the cleavage of the cell. 

Figure 1 Overview of mitosis, (a) (i) Chromosomes are replicated before mitosis, and sister chromosomes are held together, (ii) The spindle forms and chromosomes attach to spindle fibers, (iii) Chromosomes move to the center of the spindle at metaphase, (iv) Sister chromosomes separate at anaphase and move in opposite directions, (v) The cell divides as the cleavage furrow forms between the separated chromosomes, (vi) Two daughter cells form, each with exactly one copy of each chromosome, (b) Stmcture of colchicine, a small molecule that targets microtubules.

It is clear from observing chromosome movements that cell division occurs in an ordered sequence of events (Fig. la). First chromosomes attach to spindle microtubule fibers and move to the spindle equator. Only after completion of this step do sister chromosomes separate at anaphase and move to opposite sides of the cell, followed by their division into two daughter cells. Events must occur in this order for successful chromosome segregation. If the cell enters anaphase prematurely, before chromosomes have attached properly to the spindle, the sister chromosomes will not segregate equally, which leads to aneu-ploid daughter cells. Therefore, mechanisms that determine the timing of anaphase onset are critical for the success of mitosis. 

Figure 4 Correction of improper chromosome attachments by activation of Aurora kinase (45). (a) Assay schematic, (i) Treatment with the Eg5 inhibitor monastrol arrests cells in mitosis with monopolar spindles, in which sister chromosomes often are both attached to the single spindle pole, (ii) Hesperadin, an Aurora kinase inhibitor, is added as monastrol is removed. As the spindle bipolarizes with Aurora kinase inhibited, attachment errors fail to correct so that some sister chromosomes are still attached to the same pole of the bipolar spindle, (iii) Removal of hesperadin activates Aurora kinase. Incorrect attachments are destabilized by disassembling the microtubule fibers, which pulls the chromosomes to the pole, whereas correct attachments are stable, (iv) Chromosomes move from the pole to the center of the spindle as correct attachments form, (b) Structures of the Eg5 inhibitor monastrol and two Aurora kinase inhibitors, hesperadin and AKI-1. (c) Spindles were fixed after bipolarization either in the absence (i) or presence (ii) of an Aurora kinase inhibitor. Arrows indicate sister chromosomes that are both attached to the same spindle pole. Projections of multiple image planes are shown, with optical sections of boxed regions (1 and 2) to highlight attachment errors. Scale bars 5 xm. (d) After the removal of hesperadin, GFP-tubulin (top) and chromosomes (bottom) were imaged live by three-dimensional confocal fluorescence microcopy and DIC, respectively. Arrow and arrowhead show two chromosomes that move to the spindle pole (marked by circle in DIC images) as the associated kinetochore-microtubule fibers shorten and that then move to the center of the spindle. Time (minutes seconds) after the removal of hesperadin. Scale bar 5 (cm.

A network of intermediate filaments is often found as a laminating layer adjacent to a cellular membrane, where it provides mechanical support. The best example is the nuclear lamina along the inner surface of the nuclear membrane (see Figure 21-16). This supporting network is composed of lamin A and lamin C filaments cross-linked into an orthogonal lattice, which is attached by lamin B to the inner nuclear membrane through interactions with a lamin B receptor, an IFAP, in the membrane. Like the membrane skeleton of the plasma membrane, the lamin nuclear skeleton not only supports the inner nuclear membrane but also provides sites where nuclear pores and interphase chromosomes attach. Thus, the nuclear lamins organize the nuclear contents from the outside in. 

The mitotic apparatus of animal cells comprises the astral microtubules forming the asters, the polar and kine-tochore microtubules forming the football-shaped spindle, the spindle poles derived from the duplicated centrosomes, and chromosomes attached to the kinetochore microtubules (see Figure 20-31). 

Cells with abnormal numbers of chromosomes form when certain cell-cycle checkpoints are nonfunctional. As discussed in Chapter 21, the unreplicated-DNA checkpoint normally prevents entry into mitosis unless all chromosomes have completely replicated their DNA the spindle-assembly checkpoint prevents entry into anaphase unless all the replicated chromosomes attach properly to the metaphase mitotic apparatus and the chromosome-segregation checkpoint prevents exit from mitosis and cytokinesis if the chromosomes segregate improperly (see Figure 21-32, steps [H- 3D. As advances are made in Identifying the proteins that detect these abnormalities and mediate cell-cycle arrest, the molecular basis for the functional defects leading to aneuploidy in tumor cells will become clearer. 

It is clear from observing chromosome movements that cell division occurs in an ordered sequence of events (Fig. 2.1-l(a)). Chromosomes attach to spindle microtubule fibers and move to the spindle equator before sister... 

Accurate chromosome segregation in mitosis requires not only feedback control of anaphase onset but also regulation of chromosome attachment to the spindle. Each pair of replicated chromosomes must achieve a particular orientation in which microtubule fibers attach sister chromosomes to opposite poles of the spindle. Experiments in yeast showed that inhibition of the Ipll/Aurora family of kinases stabilized improper attachments [45, 46], but how the active kinase corrected attachment errors was not known. Because attachment errors are rarely observed in the presence of active Aurora kinase, this problem was particularly difficult to address. Inhibition of Aurora kinase, through experimental approaches such as genetic mutation, could be used to accumulate attachment errors, but not to examine error correction by the active kinase. Reversible small molecule Aurora kinase inhibitors present a... 

All of these issues were addressed through the development of an assay using several reversible small molecule inhibitors (Fig. 2.1-6) [44], First, treatment with the Eg5 inhibitor monastrol arrests cells in mitosis with monopolar spindles (Fig. 2.l-6(b) i). A particular chromosome attachment error in which both sisters are attached to the single spindle pole, referred to as syntelic attachment, is frequent in the monopolar spindles [49]. If monastrol... 

The microtubule cytoskeleton plays an important role in maintaining and regulating cell division. Microtubules are polymers that consist of a- and (3-tubulin heterodimers. In the process of mitosis, microtubules undergo dynamic cycles of lengthening (polymerizatiOTi) and shortening (depolymerization), and these cycles are critical for chromosome attachment to the mitotic spindle and for appropriate chromosome segregation. Thus, perturbation of microtubule dynanucs by either mechanism provides effective anticancer approaches. 

Bacteria normally divide and reproduce by asexual reproduction in a process called binary fission. Bacteria are prokaryotes, and, as such are relatively simple BU. They carry most of their genes on a single chromosome attached to the plasma membrane. When a single bacterial ceU divides, it first replicates its chromosome and attaches it to a different membrane site. When the bacterium has grown to about twice its normal size, the plasma membrane grows inward between the two chromosomes and divides the parent ceU into two daughter cells, each with a complete genome. 

It is evident that a number of unanswered questions surround the role of the nuclear membrane in DNA synthesis. Are interphase chromosomes attached at specific sites to specific areas of the nuclear membrane If so, how many of these sites correspond to regions where DNA synthesis is initiated at the start of S, and subsequently during S phase ... 

Wagenaar, E. B. 1969. End-to-end chromosome attachments in mitotic interphase and their possible significance to meiotic chromosome pairing. Chromosoma, 26 410-426. 

Spindle Mechanical Properties. How does the spindle respond to the mitotic forces so that force directionality is controlled and orderly chromosome motion results Artificial forces have been used to study chromosome attachment to the spindle. Especially informative are the centrifugation studies of Schrader (1934), Shimamura (1940), and Yamamoto (1964), and the micromanipulation experiments of Carlson (1952). Recent micromanipulation results (Nicklas and Staehly, 1967) add some details to the earlier observations and confirm them during normal prometaphase and anaphase motion. Artificial forces sufficient to stretch the chromosome cause little or no increase in the distance from the pole to the kinetochore, but there is much less resistance to lateral or poleward displacement. The spindle as a whole behaves in micromanipulation and during isolation as a single body it is a mechanical unit independent of the rest of the cell (reviewed by Mazia, 1961). The simplest, and the classic, interpretation attributes these mechanical properties to individual spindle fibers in order to account for the... 

Mitosis can now be viewed in the perspective of other chromosome motion and distribution mechanisms. The discussion here centers on the most striking contrasts afforded by recent studies of bacteria and dinoflagellates. As first argued in detail by Jacob et al. (1963) and as diagrammed in Figure 19, chromosome motion and distribution in such bacteria as Escherichia coli and Bacillus subtilis may depend upon chromosome attachment to the cell membrane (Fig. 19A), subsequent attachment of one... 

Electron microscope observations reveal that all dinoflagellates have common characteristics a vesicular cell covering, a two- to three-membrane chloroplast envelope, a persistent nuclear envelope with chromosomes attached to the envelope, and at some time in their life cycle, two dissimilar flagella (Taylor 1979 Steidinger 1983). In spite of the presence of flagella, the organisms are usually planktonic, and benthic in habit. 

After telephase each daughter cell inherits a set of chromosomes identical to that of the parent cell. This is possible because each of the parent chromosomes is duplicated and one set is distributed to each daughter cell. The cell manages this orderly distribution by providing, sometime during mitosis, an array of retractable fibers to which chromosomes attach and by... 

Microtubules are hollow cylinders which may serve as cables along which organelles and vesicles move and are positioned in the cytoplasm. They are important structural elements of cen-trioles and the mitotic spindle to which chromosomes attach during cell division. At least four tubulin-associated proteins including clathrin (Section 9.3) have been identified. 

Canard, B., and Cole, S. T., 1990, Lysogenic phages of Clostridium perfringens Mapping of the chromosomal attachment sites, FEMS Lett. 66 323-326. 

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