Nuclear Changes in Prophase

Chromosome condensation, the landmark event at the onset of prophase, often begins in isolated patches of chromatin at the nuclear periphery. Later, chromosomes condense into two threads, termed sister chromatids, which are closely paired along their entire lengths. Although chromosome condensation was first observed more than a century ago, the biochemical mechanism remains a mystery. Protein kinases are thought to drive mitotic chromosome condensation by phosphorylating a number of the hundreds of proteins associated with mitotic chromosomes. The onset of condensation correlates with phosphorylation of histones H1 by Cdk1 kinase and H3 by Aurora-B protein kinase, and both are widely used as physiological markers for mitotic cells. However, chromosome condensation still occurs when both of these phosphorylation events are blocked. Possibly, some combination of histone modifications might provide a “code” that promotes chromatin condensation (see Fig. 13-3).

Two pentameric protein complexes, condensin I and II, are major constituents of mitotic chromosomes with an essential role in chromosome architecture (see Fig. 13-19). The two complexes share a pair of ABC ATPases, SMC2, and SMC4 (structural maintenance of chromosomes) but have two different sets of three auxiliary proteins. Condensin II enters the cell nucleus during prophase, where it is required for prophase chromosome condensation. However, chromosomes of vertebrate cells that lack condensin condense rapidly once the nuclear envelope breaks down at the onset of prometaphase, so condensin is not directly responsible for mitotic chromosome condensation. Chromosomes that lack condensin separate normally at the beginning of anaphase but appear to fall apart while moving toward the spindle poles. This appears to reflect defects in their underlying structure, and condensin is required for proteins of the chromosome scaffold to assemble properly (see Fig. 13-19). The molecular explanation for these effects is not known. In vitro, condensin complexes can promote coiling and compaction of DNA, but the significance of this is also unknown.

Cytoplasmic Changes in Prophase

Most of the cytoskeleton reorganizes during prophase. Most notably, the microtubule array changes from an extensive network permeating the cytoplasm into two dense, radial arrays of short, dynamic microtubules around the duplicated centrosomes (see Chapter 34). Each of these asters eventually becomes one pole of the mitotic spindle. During prophase, the two asters usually migrate apart across the surface of the nucle-ar envelope, signaling the start of spindle assembly (Fig. 44-3).

Mitotic microtubules behave like interphase microtubules in many ways (see Chapter 34). They are nucleated at their minus ends, they grow by addition of tubulin subunits at their free plus ends, and they undergo random catastrophes during which they rapidly shorten. To a large extent, the prophase changes in microtubule organization can be explained by two simple biochemical changes: (1) increased microtubule-nucleating activity of centrosomes and (2) altered dynamic instability properties of the microtubules (Table 44-1; also see Chapter 34). Interphase microtubules have a high probability of recovering from catastrophes, so they grow quite long. Mitotic microtubules grow more rapidly but exist only transiently. This is because when they undergo a catastrophe, they usually shorten all the way back to the centrosome, with little chance of rescue. These differences in dynamic instability can be reproduced in vitro in mitotic and interphase cellular extracts. They appear to arise, at least in part, from counterbalancing interactions between microtubule-associated proteins, which promote microtubule stability, and kinesin-13 (see Fig. 36-13), which promotes microtubule disas-sembly.

Table 44-1 COMPARISON OF MICROTUBULE DYNAMICS IN INTERPHASE AND MITOTIC NEWT LUNG CELLS

* Most cellular microtubules grow constantly by addition of subunits to their free ends but occasionally stop growing and begin shrinking rapidly (a “catastrophe”). Unless shrinking is reversed (a “rescue”), the microtubule completely disappears.

Other cytoskeletal elements that disassemble during prophase include most, but not all, classes of intermediate filaments (including the nuclear lamins) and specialized actin filament structures, such as stress fibers. However, the junctional complexes between adjoining cells are maintained in epithelial cells. As a result of the cytoskeletal reorganization, most cells round up during prophase. This is particularly evident for animal cells that are cultured on a flat substrate, but cells in tissues also change their shape dramatically during mitosis.

RNA transcription of the chromosomes stops during mitosis. Although a number of mechanisms contribute to this change, phosphorylation of components of the transcriptional machinery by Cdk1 kinase appears to be the predominant mechanism. Cdk1 kinase phosphorylation of ribosomal elongation factor EF2a also stops ongoing protein synthesis and assembly of new ribosomes. Phosphorylation of several nucleolar proteins leads to disassembly of the nucleolus.

The Golgi apparatus and endoplasmic reticulum fragment or vesiculate during prophase (Fig. 44-4). In ad-dition, many membrane-mediated events, including fluid-phase pinocytosis, endocytosis, exocytosis, and intracellular sorting of membrane components (see Chapters 21 and 22), greatly decrease. Golgi disassembly is driven by several kinases, including Cdk1. The first step of the process is fragmentation of the Golgi into smaller mini-stacks. The second step is still being investigated. Some evidence argues that Cdk1 phosphorylation of key components prevents the fusion of transport vesicles back into Golgi stacks (see Chapter 21), the net result being that the Golgi buds apart into small vesicles. Other evidence suggests that an imbalance of vesicle flow between the Golgi and the endoplasmic reticulum results in the Golgi being absorbed into the ER during mitosis. Whatever the mechanism of its disassembly, Golgi reassembly begins again during anaphase, following inactivation of Cdk1 kinase. Some Golgi derived vesicles contribute to the plasma membrane during cleavage furrow ingression at the end of mitosis (see later).

Figure 44-4 golgi apparatus dynamics in interphase and mitosis. Disassembly in mitosis is driven by phosphorylation of components blocking fusion of Golgi membranes.

Nuclear Envelope Disassembly in Prometaphase

Nuclear envelope disassembly involves the removal of two membrane bilayers coupled with disassembly of the nuclear pores and the fibrous nuclear lamina meshwork that underlies the inner bilayer (Fig. 44-6). Protein phosphorylation triggers breakdown of the nuclear envelope but the critical targets and mechanisms are not entirely known. Phosphorylation of the nuclear lamins at two sites flanking the central coiled-coil causes the lamina network to disassemble into subunits and might contribute to disassembly of the envelope. Cdk1 kinase can phosphorylate these lamin residues in vitro, but other kinases might participate in vivo. Additionally, phosphorylation of nucleoporins leads to nuclear pore disassembly. This fenestrates the nuclear envelope, dissolving the barrier between nucleus and cytoplasm. Interaction between microtubules and dynein associated with the nuclear envelope may also rip holes in the envelope.

Figure 44-6 disassembly of the nuclear envelope during mitosis. a– B, Two contrasting models explaining the fate of the nuclear envelope during the transition from interphase to mitosis in a higher eukaryote. C, Micrographs showing solubilization of lamin A fused to GFP during mitosis. Scale bar is 10μm. D–E, Reversible disassembly of lamins A, C, and B is driven by posttranslational modifications of the lamin polypeptides.

(C, Courtesy of William C. Earnshaw.)

Nuclear envelope components are dispersed in the cytoplasm from prometaphase until telophase (Fig. 44-6), but the mechanism may differ in various cell types. In fertilized amphibian eggs, the nuclear membrane breaks up into small vesicles that disperse in the cytoplasm. In vertebrate somatic cells, the nuclear envelope may be absorbed into the endoplasmic reticulum, which remains as an extensive tubular network throughout mitosis. In both cases, lamin B remains associated with the dispersed nuclear envelope, whereas lamins A and C and many proteins of the nuclear pore complexes disperse as soluble subunits.

During prophase, kinetochores transform from nondescript balls of condensed chromatin into organized plaques on the surface of the chromosomes. By early prometaphase, the characteristic trilaminar disk structure (see Fig. 13-20) can be seen. Each sister chromatid has a kinetochore. Thus, sister kinetochores are located on opposite faces of the mitotic chromosome. Only one of the two sister kinetochores faces a given spindle pole at any one time.

Organization of the Mitotic Spindle

The mature metaphase spindle is a bilaterally symmetrical structure with centrally located chromosomes flanked by arrays of microtubules radiating from the poles (Fig. 44-7).

Figure 44-7 role of motors in spindle structure. The mitotic spindle is a dynamic entity whose structure depends on microtubule assembly/disassembly plus the balance of forces that act to slide microtubules relative to one another and to pull the poles together or apart. A, In metaphase, the structure is at steady state. The forces that tend to elongate the spindle, including cytoplasmic dynein (which moves toward microtubule minus ends, pulling the poles out toward the cell cortex) and bipolar kinesin-5 (which moves toward microtubule plus ends, pushing the poles apart), are counterbalanced by kinesin-14, which moves toward microtubule minus ends (and pulls the poles together) and microtubule disassembly at the spindle poles. Dynein and its associated protein, NuMA, also have an important role in organization of the spindle pole. B, In anaphase, the balance of kinesin activity shifts, microtubule disassembly at the poles declines, and the spindle undergoes a dramatic elongation. During anaphase, bipolar kinesin-5 and PRC1 also have important roles in organizing the central spindle, which is essential for subsequent assembly and function of the cleavage furrow.

Three predominant classes of microtubules are present in the metaphase spindle (Fig. 44-12). Kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at or near the spindle pole. They characteristically form bundles, called kinetochore fibers, which contain anywhere from 1 microtubule in the budding yeast to more than 200 microtubules in some higher plants. Each human kinetochore binds about 20 microtubules. Up to about 80% of the approximately 2200 spindle microtubules in humans may be present in kinetochore fibers. Interpolar microtubules are distributed throughout the body of the spindle and do not attach to kinetochores. Their minus ends may terminate near the pole but are not physically linked to it so that they appear to be free at both ends. Many interpolar microtubules penetrate between and through the chromosomes and extend for some distance beyond them. Thus, the central spindle contains a large number of interdigitated antiparallel microtubules. Tracking these spindle microtubules by electron microscopy has revealed a tendency for the interdigitated microtubules of opposite polarity to pack next to one another. During late anaphase, these antiparallel microtubules bundle to form a structure, called the central spindle, that appears to have important roles during cytokinesis. Astral microtubules project out from the poles and have a role in orienting the spindle in the cell through interactions with the cell cortex. All of the microtubules within each aster have the same polarity, with their minus ends proximal to the pole. Each unit of a spindle pole, with its associated kinetochore and interpolar and astral microtubules, is referred to as a half-spindle.

Figure 44-12 introduction to metaphase. A, Summary of the major events of metaphase. B, Distribution of DNA (blue), microtubules (red), actin (green), and gamma tubulin (centrosomes [yellow]) in a metaphase PtK1 (rat kangaroo) cell.

(B, Courtesy of Dr. Alexey Khodjakov, Wadsworth Center, Albany, New York.)

Spindle structure is largely determined by a combination of microtubule dynamics plus the action of at least seven different types of kinesins plus cytoplasmic dynein (see Chapter 36). These motors often work in opposition to one another. As a result, the spindle is a highly dynamic structure whose morphology changes as the balance of forces shifts between the various motors. For example, inactivating one or more kinesins with drugs or switching a temperature-sensitive mutant to the nonpermissive temperature can cause the spindle to collapse rapidly on itself. Consequently, chromosomal movements and changes in spindle morphology are complex processes that reflect both the dynamic growth and shrinkage of microtubules plus the net vectorial output of multiple antagonistic and synergistic motors. These various components interact; for example, force exerted by motors can influence the dynamic assembly/disassembly of microtubules.

Spindle Assembly

In metazoans, spindle assembly starts in prophase with the separation of the asters. In most cells, each aster is organized around a centrosome, consisting of a centriole pair and associated pericentriolar material. γ-Tubulin ring complexes in the pericentriolar material efficiently nucleate microtubules (see Fig. 34-16), so each aster acts as a microtubule organizing center. By the end of prophase, the spindle consists of two asters linked by a few interpolar microtubules. Cytoplasmic dynein at the cell cortex exerts an outward force separating the asters, whereas kinesin-14 motors (which move toward microtubule minus ends) on the interpolar microtubules exert a counterbalancing force holding the asters together.

This balance of forces changes when the nuclear envelope breaks down. Bipolar kinesin-5 motors are phosphorylated by Cdk1 kinase and concentrate in the central spindle, where they cross-link adjacent antiparallel interpolar microtubules. Kinesin-5 moves toward the plus ends of microtubules. If such a motor attaches to two adjacent antiparallel microtubules and begins to move, it will cause them to slide apart (Fig. 44-7). Thus, the action of kinesin-5 motors pushes the spindle poles apart. The two half-spindles do not separate because they are physically linked via the chromosomes, with sister kinetochores attached to opposite spindle poles.

Also at this time, the asters mature into focused spindle poles. The pericentriolar material efficiently nucleates the assembly of new microtubules with their minus ends at the pole. In addition, cytoplasmic dynein transports free microtubules that are nucleated throughout the cytoplasm to the centrosome along astral microtubules for incorporation into the spindle. The focused microtubule array at the pole forms partly owing to the tethering of microtubules by centrosomes, and partly due to the concerted action of various motors and microtubule cross-linking proteins such as nuclear mitotic apparatus protein (NuMA). NuMA is released from the nucleus on nuclear envelope breakdown, and it accumulates near the poles at the minus ends of microtubules.

In large cells that lack centrosomes, such as eggs, spindle formation depends on an alternative pathway that is also active in cells with centrosomes (Fig. 44-8). Chromosomes stabilize nearby microtubules, which are then organized into a bipolar spindle by motor proteins and NuMA. This spindle assembly pathway involves importin a and b, two proteins that direct traffic through nuclear pores during interphase (see Fig. 16-14). Importin a and b inhibit mitotic spindle formation by sequestering several essential proteins, including NuMA. Chromosomes counteract this by releasing spindle assembly factors such as NuMA from importin a and b. The mechanism depends on the association of the GTP exchange factor RCC1 (Ran-GEF in Fig. 44-17) with chromosomes. RCC1 produces a local gradient of the active GTPase Ran-GTP, which dissociates NuMA from importin a and b just as it does during protein import into the nucleus. In this case, however, the net result is microtubule stabilization and assembly of the mitotic spindle.

Figure 44-8 assembly of a bipolar spindle in the absence of centrosomes. A gradient of Ran-GTP stabilizes microtubules around chromosomes, releasing spindle assembly factors. Microtubules that accumulate around the chromosomes are sorted and organized by motor proteins and are subsequently focused to make poles by motors and (–) end-binding proteins such as NuMA. These spindle poles lack prominent astral microtubules.

Figure 44-17 chromosomes move on shrinking microtubules during anaphase. A, Mitotic cells are injected with a fluorescently labeled tubulin that rapidly becomes incorporated into the spindle. Just after anaphase onset, a laser is used to photobleach a stripe (white) across the spindle near the upper pole. The live cell is monitored over time by fluorescence (B) and phase contrast (C) microscopy. In this mammalian cell, the chromosomes approach the bleached stripe much faster than the stripe approaches the spindle pole. In other organisms with higher rates of microtubule flux in their spindles, the bleached zone would also move appreciably toward the pole. The numbers are time in seconds.

(B–C, Reproduced from Gorbsky GJ, Sammak PJ, Borisy GG: Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol 106:1185–1192, 1988. Copyright 1988 The Rockefeller University Press.)

If centrosomes are removed or destroyed experimentally, somatic cells can also use motor proteins to organize microtubules into bipolar spindles that lack asters but are otherwise remarkably normal. However, only about half of the cells that have lost centrosomes manage to complete mitosis successfully. Thus, centrosomes are not required to form spindles, but they do contribute to successful chromatid separation and cytokinesis by helping to orient the spindle within the dividing cell.

Chromosome Attachment to the Spindle

Dynamic microtubules of prometaphase asters scan the cytoplasm searching for both binding sites that will capture and stabilize their distal plus ends and for other components, including free microtubules. Breakdown of the nuclear envelope makes the condensed chromosomes accessible to the microtubules. Chance encounters with kinetochores during cycles of growth result in the plus ends of microtubules being captured by the kinetochore. Capture probably involves the nine-component KMN complex, which contains two components capable of binding weakly to microtubules. One of these, the helical Ndc80 complex (see Fig. 13-21) binds along the sides of microtubules forming fine hairs visible in the electron microscope. Other members of the complex provide an anchoring site for Ncd80 in the kinetochore. Captured microtubules are about five-fold less likely to depolymerize catastrophically than free microtubules. When catastrophes do occur, the microtubules depolymerize back to the pole, recycling tubu-lin subunits for incorporation into other, growing microtubules.

Initial attachment of a chromosome to a microtubule often involves a lateral interaction between the corona region of the kinetochore (see Fig. 13-20) and the side of a microtubule (Fig. 44-9). Cytoplasmic dynein then slides the chromosome rapidly along the microtubule toward the pole. These steps were first seen in animal cells, and a similar pattern of chromosome attachment and movement also occurs in budding yeast cells.

Figure 44-9 initial chromosomal movements during prometaphase. a–b, Chromosomal movements initiate with the capture of a microtubule by the kinetochore. This results first in movement toward the pole from which that microtubule originated. These images come from a study in which living cells, observed by differential interference microscopy, were subjected to rapid chemical fixation just after a chromosome had attached to the spindle (arrow). C, Attachment of the chromosome to the spindle was confirmed by indirect immunofluorescence staining for tubulin and, ultimately, by thin-section electron microscopy (D). E, The graph shows the movements of the chromosomes before and after attachment.

(Reproduced from Rieder CL, Alexander SP, Rupp G: Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol 110:81–95, 1990. Copyright 1990 The Rockefeller University Press.)

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