Mitosis

Abstract

SUMMARYAll eukaryotic cells prepare for cell division by forming a "mitotic spindle"-a bipolar machine made from microtubules (MTs) and many associated proteins. This device organizes the already duplicated DNA so one copy of each chromosome attaches to each end of the spindle. Both formation and function of the spindle require controlled MT dynamics, as well as the actions of multiple motor enzymes. Spindle-driven motions separate the duplicated chromosomes into two distinct sets that are then moved toward opposite ends of the cell. The two cells that subsequently form by cytokinesis, therefore, contain all the genes needed to grow and divide again.

Figure 1.

The cellular growth and division cycle. (A) Cartoon of the main segments of the cell cycle. During interphase (G₁, S, G₂), the cell accomplishes sufficient biosynthesis to become two. In mitosis (M), cell parts are reorganized so the mitotic spindle can achieve the equipartition of the chromosomes and centrosomes, leaving the distribution of more numerous components, such as ribosomes, to the laws relating to large numbers and the process of cytokinesis (C), in which the cell itself divides into two daughters. In tissues, cells can continue further rounds of division or can exit the cell cycle. (B) Chromosome behavior during the cell cycle. During the first gap phase, G₁, cells have only the chromosome they were given at the previous cell division; each decondensed chromosome is a single DNA duplex. This phase is followed by a period of chromosome replication in S phase, and then a further gap phase, G₂, in which the newly replicated sister chromatids are held together by cohesins. During mitosis, the condensed sister chromatids are separated in a process highly dependent on interactions between microtubules (MTs) and chromosomal kinetochores. (C–F) Immunofluorescence images of mitotic cells, sourced from the mammalian rat kangaroo PtK₁ strain. In prophase (C), the chromosomes (blue) condense inside a still-patent nucleus while MTs (green) organize in the cytoplasm. In prometaphase (D), the spindle MTs (red) gain access to the chromosomes (blue) and attach to the kinetochores (yellow) that will subsequently govern most chromosome motions. By metaphase (E), the chromosomes are quite accurately aligned on the spindle midplane. During anaphase (F), they segregate, moving toward opposite poles of the spindle, and the spindle itself elongates. Scale bars, 2 µm. (A,B, Reprinted, with permission, from McIntosh et al. 2012; C–F, previously unpublished micrographs, kindly provided by Jennifer DeLuca, Department of Biochemistry and Molecular Biology, Colorado State University.)

Figure 2.

Electron micrographs of cells as they form a mitotic spindle. Rat kangaroo PtK1 cells were cultured on gold grids coated with a thin layer of plastic and carbon, lysed with 0.2% Triton X100 and 1 mm MgCl2, in a PIPES-HEPES buffer, pH 7.2, and then fixed with 2% paraformaldehyde and 0.1% glutaraldehyde. These samples were quenched in 0.2 mg/mL NaBH₄ in 1:1 ethanol:phosphate-buffered saline, then stained with a monoclonal antibody against tubulin, followed by a rabbit antimouse IgG bound to 10-nm colloidal gold, followed by fixation in osmium tetroxide, and then by drying with the critical-point method. Microtubules (MTs) in the interphase and mitotic cells are nicely contrasted, and the chromosomes are stained by osmium. (A) Interphase: The nucleus (N) contains decondensed chromatin. Intermediate filaments (IFs) surround the nucleus. (B) Early prometaphase: CHRs, chromosomes; C, centrosome. (C) Late prometaphase: AMTs, astral microtubules. (D) Metaphase: KMTs, kinetochore microtubules. Scale bars, 1 µm. (Images kindly provided by Mary Morphew, University of Colorado, Boulder.)

Figure 3.

Formation of a closed mitotic spindle occurs within the nucleus. Slice from an electron tomogram of a dividing budding yeast cell during prometaphase. The nuclear envelope (NE), the spindle pole bodies (SPBs), and the microtubules are clear, but, in this cell type, chromosome condensation is not sufficient to make the chromatin obvious. Scale bar, 1 µm. (Image kindly provided by Eileen O’Toole, University of Colorado, Boulder; reprinted, with permission, from McIntosh et al. 2012.)

Figure 4.

Structure of the kinetochore. (A) Slice from an electron tomogram of a rat kangaroo PtK₁ cell in prometaphase. A chromosome (C) and the associated microtubules (MTs) are easy to distinguish in the tomogram; their point of connection is the kinetochore (arrow). (B) Diagram of kinetochore composition and structure. At mitotic entry, phosphorylation (P) by activated CDK–cyclin-B promotes assembly of the outer kinetochore on a platform of constitutive kinetochore proteins. For information about the kinetochore proteins shown here, see Cheeseman 2014. CDK, cyclin-dependent kinase. (Reprinted, with permission, from Cheeseman 2014, © Cold Spring Harbor Laboratory Press.)

Figure 5.

Rat kangaroo PtK₁ cell with separating chromosomes. Cultured cells were lysed and fixed as for Figure 2 and then embedded, sectioned, and imaged on a high-voltage electron microscope. Arrows labeled 1 and 2 indicate initial and secondary sites, respectively, of kinetochore microtubule (KMT) depolymerization during anaphase A. CHRs, chromosomes. Scale bar, 1 µm.

Figure 6.

Anaphase B. Rat kangaroo PtK1 cells fixed at mid (A) and late (B) stages of spindle elongation and prepared as in Figure 2. ipMTs are the interpolar microtubules, which interdigitate at the spindle midplane. These elongate by sliding and the addition of tubulin at the sites indicated by white arrows. CF, cleavage furrow; CHRs, chromosomes. Scale bars, 1 µm. (Images kindly provided by Mary Morphew, University of Colorado, Boulder.)