Mitosis and Meiosis Part A

Luísa T. Ferreira , ... Helder Maiato , in Methods in Cell Biology, 2018

3.5.4 Calculating microtubule turnover rates

1.

Align spindle poles horizontally and generate whole-spindle, sum-projected kymographs (sum projections generated using ImageJ and kymographs generated as previously described in Pereira & Maiato, 2010).

2.

Quantify fluorescent intensities for the PA/PC spindle region for each time point manually (or using a custom-written routine in MATLAB) and normalize intensities to the first time point after PA/PC following background subtraction (background values obtained from quantifying the nonactivated other half-spindle).

3.

Correct values for photobleaching by normalizing to the values obtained from the quantification of fluorescence loss of whole cell (including cytoplasm), sum projected images (i.e., each cell has its own bleaching constant). Alternatively, normalize to averages obtained from the quantification of fluorescence loss of defined spindle regions of cells treated with 1–5   μM Taxol to fully stabilize MTs (Orr et al., 2016). For each of these methods, do not use background-subtracted values, since these expose nonlinear photobleaching kinetics that may be observed during the first 1–2   min of imaging.

4.

To calculate MT turnover, fit the normalized intensity values at each time point (corrected for photobleaching) to a double exponential curve A1   ×   exp(−  k 1  × t)   + A2   ×   exp(−  k 2  × t) using MATLAB (Mathworks), in which t is time, A1 represents the less stable (ipMTs) population, and A2 the more stable (kMT) population with decay rates of k 1 and k 2, respectively (avoid using cells that display an R 2 value <   0.99).

5.

From these curves, obtain the rate constants and the percentage of MTs for the fast (typically interpreted as the fraction corresponding ipMTs) and the slow (typically interpreted as the fraction corresponding K-MTs) processes.

6.

The half-life is calculated as ln   2/k for each population of microtubules.

7.

Use the Student's two-tailed t-test to perform statistical analysis on the results and make sure to discriminate between prometaphase and metaphase cells (that have different MT dynamics) (Fig. 7).

Fig. 7

Fig. 7. Calculating MT turnover through photoactivation. (A) DIC and time-lapse fluorescent images of a representative metaphase U2OS cell expressing PA-GFP-α-tubulin and mCherry-α-tubulin. The mitotic spindle is visualized by mCherry fluorescence. Fluorescent images are inverted for better visualization of the photoactivated GFP molecules. (B) Sum-projected, whole-spindle kymograph generated to quantify the fluorescence dissipation after photoactivation (FDAPA). Dashed white lines indicate the spindle poles; yellow lines indicate the boundaries used to quantify the signal generated from PA; and red lines indicate the boundaries used for determining background levels. (C) Normalized fluorescence intensity. Once fitted as a double exponential curve, the values obtained allow for the calculation of the dynamics of fast and slow MT populations. (D). Photoactivation troubleshooting flowchart used to determine the exclusion criteria for calculating MT turnover using PA.

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Cell Motility and Behavior

Wallace F. Marshall , in The Chlamydomonas Sourcebook, 2009

C. Mitotic spindle poles

The organization of the mitotic spindle pole in Chlamydomonas differs from that of animal cells in terms of the location of the centrioles. In contrast to animal cells where centrioles are buried in the center of the mitotic spindle pole, electron microscopic studies in Chlamydomonas have clearly shown that the basal bodies are not located directly at the spindle poles (Johnson and Porter, 1968; Coss, 1974). Instead, the basal bodies maintain an attachment to the poles via centrin-containing fibers known as nucleus-basal body connectors (NBBCs) (Wright et al., 1985). These fibers, defined based on immunofluorescence staining with anti-centrin antibodies, appear similar to fibers known as "rhizoplasts" (Kater, 1929), and indeed centrin was first identified as a protein obtained from isolated rhizoplasts of the green alga Tetraselmis (Salisbury et al., 1984). Yet another term sometimes used in the literature to describe this structure is "striated rootlet." The NBBC is dispensable for mitosis, as mutants lacking this structure can perform normal mitosis (Wright et al., 1989).

The situation has been clarified by electron microscopy studies, conducted in the related unicellular green alga Spermatozopsis (Lechtreck and Grunow, 1999), in which images of different stages of early mitosis indicate that spindle microtubules are initially nucleated near the base of the basal body, which migrates to the nuclear envelope early in mitosis. As the nuclear envelope becomes fenestrated, the microtubule cloud surrounding the basal body is deposited on the nuclear surface, and the basal body then separates from the pole but remains tethered to it by distinct fibers, presumably composed of centrin. It is likely that a similar situation will hold in Chlamydomonas.

Mutants in the centrin protein VFL2 show a loss of basal bodies at the spindle poles. vfl2 mutants also show an increased rate of mitotic chromosome loss (Zamora and Marshall, 2005). This may indicate that spindle poles lacking basal bodies function less effectively in mitotic chromosome segregation; alternatively, this result might reflect some basal body-independent function for centrin.

If basal bodies are not actually within the spindle pole, this leaves us to ask what composes the spindle pole itself. Gamma-tubulin is present in Chlamydomonas and localizes to the basal bodies and can also be seen at the poles of the spindle (Dibbayawan et al., 1995; Silflow et al., 1999), suggesting that the basic microtubule-nucleating function of the spindle pole in Chlamydomonas will be similar to that of other organisms. The Chlamydomonas genome encodes clear homologues of the gamma-tubulin ring complex proteins GCP2 (EDP00586), GCP3 (EDO98538), and GCP4 (EDP09233), suggesting that the basic molecular organization of the gamma-tubulin ring complex is conserved in Chlamydomonas. It is more difficult to say whether or not the overall organization of the centrosome is conserved with animal cells, since the main pericentriolar proteins such as pericentrin contain sufficient coiled-coil and other simple sequence to make it very hard to identify homologues in other eukaryotes.

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A Survey of Cell Biology

Claire E. Walczak , Rebecca Heald , in International Review of Cytology, 2008

5.2 Anaphase B spindle pole separation

The driving apart of the spindle poles during anaphase B is thought to be accomplished by forces within the spindle as well as with forces on the astral microtubules where they contact the cortex ( Rosenblatt, 2005). As chromosomes segregate, they leave behind "passenger proteins" at the equator, many new factors are recruited, and a microtubule structure called the central spindle forms. Motors that slide antiparallel microtubules in the central spindle are thought to make a major contribution to anaphase B. In Drosophila embryos, it has been proposed that the kinesin-5 Klp61F drives the sliding of antiparallel microtubules while kinesin-13 Klp10A drives depolymerization at poles and that the kinesin-4 Klp3A suppresses flux to couple sliding to spindle elongation (Brust-Mascher et al., 2004; Brust-Mascher and Scholey, 2002). In addition, members of the kinesin-6 family, including MKLP1, may drive microtubule sliding through organization of the microtubule bundles in the central spindle. Inhibition of MKLP1 by antibody injection caused a block in mitotic progression and disorganized central spindles (Matuliene and Kuriyama, 2002, 2004; Nislow et al., 1990, 1992). More recently, RNAi knockdown showed that the kinesin-6 proteins MKLP1 and MKLP2 are critical for central spindle organization that is necessary for cytokinesis (Zhu et al., 2005). Furthermore, laser microsurgery experiments in yeast revealed that forces for sliding of the central spindle are indeed able to drive spindle pole separation (Khodjakov et al., 2004). Together, these data support the idea that motor proteins can cross-link and slide microtubules in the central spindle to drive spindle pole separation. However, it is also clear that motors organize the central spindle late in mitosis, and their inhibition gives rise to cytokinesis defects. It therefore has yet to be determined which motors are essential for antiparallel microtubule sliding during anaphase B in vertebrate cells, a clearly important area of future research.

It is also thought that interactions of microtubules with the cortex contribute to anaphase spindle pole separation (Rosenblatt, 2005). The experiments looking at how inhibition of the interaction of microtubules with the cortex have been problematic because the proteins involved, such as cytoplasmic dynein, also appear to function in the initial stages of centrosome separation at prophase (Vaisberg et al., 1993). This raises the general issue that high temporal resolution is necessary to inhibit protein function in anaphase without disturbing the system earlier, which could cause secondary defects. The development of reagents such as fast-acting small molecule inhibitors that could disrupt central spindle components, or cortical microtubule interactions specifically during anaphase B is essential to address the mechanisms driving anaphase B in vertebrate cells.

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Microtubules, in Vitro

Anutosh Ganguly , ... Fernando Cabral , in Methods in Cell Biology, 2013

Summary

Microtubule detachment from centrosomes and spindle poles is a poorly understood process whose importance is only now becoming appreciated. It was initially proposed to play a role in cytoplasmic microtubule turnover ( Keating et al., 1997), but more recent studies have demonstrated that it is a cell cycle-regulated process that plays a critical role in cell division and in the mechanism of action of drugs that affect mitotic spindle formation (Ganguly & Cabral, 2011). These drugs either stimulate (e.g., colchicine, vinblastine, nocodazole) or suppress (e.g., paclitaxel, epothilones) microtubule detachment indicating that the frequency of detachment must be maintained at appropriate levels for normal mitotic progression. Tubulin mutations that interfere with cell division often act by stimulating microtubule detachment, and their effects can be counteracted by depletion of MCAK (Ganguly, Yang, Pedroza, et al., 2011). Similarly, toxic effects due to overexpression of MCAK can be counteracted by treatment with paclitaxel (Ganguly, Yang, Pedroza, et al., 2011). Hence, it is likely that kinesin-related proteins such as MCAK play a significant role in the mechanism of detachment, and future studies will be needed to determine what other proteins are involved in the mechanism and how those proteins are arranged and regulated. More work will also be needed to determine how the microtubules are released and the role that microtubule detachment plays during cell division. Is detachment necessary to remodel the interphase microtubules when cells enter mitosis? Does it provide microtubule fragments for mitotic spindle assembly? Does it occur continuously throughout mitosis or only at specific stages? Attempts to answer these and related questions will provide exciting challenges for years to come.

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Essays on Developmental Biology, Part A

Aaron F. Severson , ... Bruce Bowerman , in Current Topics in Developmental Biology, 2016

10 A Pushy View of Kinetochore Function and Chromosome Dynamics During Oocyte Meiosis

The influence of microtubule–kinetochore attachment on oocyte spindle pole coalescence in C. elegans brings us to an intriguing controversy regarding the importance of kinetochores during oocyte meiosis (Fig. 4B and C). An important early observation was that depleting either components of the Ndc80 complex, responsible for the microtubule attachment activity of the kinetochore, or core kinetochore components such as KNL-1, had remarkably minor effects on meiotic spindle assembly and chromosome organization (Dumont et al., 2010). In live-imaging studies of microtubule and chromosome dynamics, using fusions of GFP and mCherry to β-tubulin and a histone, respectively, kinetochore disruption was shown to cause only minor perturbations in spindle morphology, chromosome congression to the metaphase plate, and anaphase chromosome segregation to the poles. During meiosis I in wild-type C. elegans oocytes, spindles shorten substantially prior to anaphase, and during anaphase the poles rapidly disassemble and most spindle microtubules are detected between the separating chromosomes (Fig. 4B). Moreover, knockdown of factors required for the assembly or stability of these anaphase microtubules, such as the microtubule-stabilizing protein CLS-2/CLASP, resulted in both a substantial loss of the interchromosomal microtubules and severe defects in anaphase chromosome movements (Dumont et al., 2010). These findings led Dumont and Desai to propose a model in which microtubule polymerization between the segregating chromosomes pushes the chromosomes apart during anaphase of meiosis I and II, with little if any requirement for microtubule–kinetochore interactions and poleward pulling forces during anaphase (Fig. 4B).

Whether the apparent lack of a substantial role for kinetochores during anaphase in C. elegans oocytes is relevant to other species is not known. However, TEM analysis of fixed mouse oocytes during meiosis I and II clearly indicate that microtubules attach to kinetochores, and the kinetochore regions lead the anaphase movements of meiotic chromosomes toward the poles (Brunet et al., 1999). Microtubule–kinetochore attachments have also been observed in human oocytes, although the correction of improper syntelic and merotelic attachments appears to be remarkably inefficient (Holubcova et al., 2015). Furthermore, knockdowns of Ndc80 complex components in mouse oocytes cause substantial defects in spindle organization and chromosome segregation (Sun et al., 2010, 2011), and perturbations of mouse meiotic spindle assembly lead to activation of the kinetochore-based spindle assembly checkpoint (Ma et al., 2010; McGuinness et al., 2009). Nevertheless, very few microtubule–kinetochore attachments form prior to metaphase of meiosis I in mouse oocytes and microtubule–kinetochore attachments may not be required for chromosome congression to the metaphase plate (Brunet et al., 1999). Although mutational inactivation of γ-tubulin disrupts microtubule–kinetochore attachments in Drosophila oocytes, the role of these attachments in chromosome segregation is unknown (Hughes et al., 2011). Systematic investigations of how microtubule–kinetochore attachments, and kinetochore function more generally, influence spindle assembly and chromosome movement are needed to fully assess and compare the role of these structures during oocyte meiotic cell division in different animal species.

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Mitosis

P. Wadsworth , J. Titus , in Encyclopedia of Biological Chemistry (Second Edition), 2013

Anaphase

During anaphase, sister chromatids separate and move to the spindle poles ( Figures 2 and 3 ). Anaphase consists of two phases, anaphase A and B. During anaphase A, the chromosomes move to the poles and kinetochore fiber microtubules shorten; during anaphase B, the spindle poles move apart as interpolar microtubules elongate and slide past one another. Many cells undergo both anaphase A and B motions, but, in some cases, one or the other motion dominates.

Separation of the paired sister chromatids is required for poleward motion in anaphase. Chromatid separation results from the proteolytic degradation of components that link the chromatids at the centromere. Degradation is triggered by the activity of the anaphase-promoting complex, which regulates cell-cycle progression. Chromatid separation is not the result of tugging by microtubules and motor proteins, and can be observed even in the absence of microtubules.

Although the motion of the chromosomes to the spindle poles in anaphase has fascinated biologists for many years, the molecular basis for this motion remains controversial and incompletely understood. During anaphase A, kinetochore microtubules must shorten as the chromosomes move poleward. Measurements of spindle flux show that subunit loss from microtubules occurs at the spindle poles during anaphase. In many cells, however, the rate that chromosomes move exceeds the rate of subunit loss at the pole, and, thus, subunit loss must also occur at the kinetochore.

Pioneering studies of mitosis in living embryonic cells demonstrated that assembly and disassembly of microtubule polymers result in chromosome motion. This work led to the hypothesis that microtubule disassembly drives chromosome motion. Later work identified molecular motors at the kinetochore, leading to the alternative hypothesis that forces generated by molecular motors drive chromosome motion. One possibility is that molecular motors power chromosome motion, but kinetochore microtubule disassembly limits the rate of chromosome motion. Alternatively, disassembly may be responsible for chromosome motion, and motors may tether the chromosomes to the shortening fiber. The presence of potentially redundant mechanisms for chromosome motion may reflect the fact that mitotic fidelity is of utmost importance.

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A Survey of Cell Biology

James R. Aist , in International Review of Cytology, 2002

E Anaphase B

Anaphase B begins when all the chromatids have reached the spindle poles ( Inoué and Ritter, 1975), and it ends, by definition (Bayles et al., 1993), when the central spindle has been fully extended and is no longer continuous from pole to pole. This is the mitotic stage during which the central spindle elongates rapidly and considerably, up to three or four times its metaphase length in Fusarium spp. (Aist, 1969; Aist and Bayles, 1988). In F. solani f. sp. pisi, the rate of spindle elongation (as measured in living cells) is an almost unprecedented 6 or 7 μm/min (Aist and Bayles, 1988). By comparison, the rate in fission yeast is 1.4 or 1.5 μm/min (Hagan et al., 1990; Nabeshima et al., 1998) and in budding yeast the fast phase occurs at nearly the same rate as in fission yeast (Kahana et al., 1995; Saunders et al., 1995; Yeh et al., 1995). Anaphase B takes slightly more than 1   min in F. oxysporum (Aist and Williams, 1972) and about 2   min in F. solani f. sp. pisi (Aist and Bayles, 1988). Typically, the anaphase B nucleus in Fusarium spp. oscillates back and forth within the cell (Aist, 1969; Aist and Bayles, 1988), which was one of the first indications that the mitotic apparatus may be pulled on during this stage by an extranuclear force acting on the SPBs via astral MTs.

The nuclear envelope becomes greatly attenuated during anaphase B by forces that elongate the spindle because it does not break down until late in this stage (Aist, 1969; Aist and Berns, 1981; Aist and Williams, 1972). Laser trapping experiments (Berns et al., 1992) have demonstrated the amazing elasticity of the nuclear envelope in living cells of F. solani f. sp. pisi. As the spindle is elongated, the subterminal portions of the original, now attenuated, nuclear envelope pinch down behind the incipient daughter nuclei (Aist, 1969; Aist and Berns, 1981; Aist and Williams, 1972); thus, the nuclear envelopes surrounding the daughter nuclei are derived directly from the corresponding ends of the original nuclear envelope during anaphase B. In late anaphase B, the remnant nuclear envelope between the daughter nuclei disintegrates and disappears from view in living cells (Aist, 1969; Aist and Bayles, 1988).

Only recently have mitotic asters garnered much attention in the search for mitotic mechanisms in any major group of organisms. Even after it was established that mitotic fungal nuclei have fairly well-developed asters, almost everyone assumed that forces developed within the spindle were solely responsible for separation of the daughter genomes during anaphase B (Aist and Morris, 1999). Consequently, most of the descriptive research on mitosis in fungi virtually ignored the mitotic asters, even until the mid-1990s (Ding et al., 1993; Winey et al., 1995). Documentation of the mitotic asters in Fusarium spp. began in the early 1970s when Aist and Williams (1972) reported that the mitotic apparatus in chemically fixed hyphae of F. oxysporum included microtubular asters that were developing during anaphase A, became maximally developed during anaphase B, and diminished to the minimal interphase state during telophase.

Mitotic asters of F. solani f. sp. pisi have been extensively documented and analyzed in living cells (Aist and Bayles, 1991d) and in cells prepared by freeze substitution (Aist and Bayles, 1991a), a method that greatly improves the preservation of organelles, especially the MTs (Howard and Aist, 1979; Howard and O'Donnell, 1987). In living cells, the asters cannot be seen directly, but directed movements of organelles within the asters toward and from the SPB reveal the shape and extent of the astral region (Aist and Bayles, 1991d). The movements of organelles in the astral region occur about equally in both directions, such that little or no buildup of organelles occurs in the aster, in contrast to the marked accumulation of organelles in asters of animal cells (Rebhun, 1972) and in those of a basidiomycete fungus (Girbardt, 1968). Electron microscopy has provided evidence that membrane-bounded organelles within the asters of F. solani f. sp. pisi are cross-bridged to the astral MTs (Aist and Bayles, 1991d). Such cross-bridging could represent the structural manifestation of the MT-associated motor proteins that would be expected to generate the mechanical force to move the organelles within the asters. Moreover, it is possible that a mechanical force that would draw the tip of a mitochondrion toward the SPB at anaphase B would be offset by a corresponding mechanical force in the opposite direction since the entire mitochondria do not get pulled into the aster but rather seem to simply become stretched out in the direction of the SPB and then spring back suddenly in the opposite direction (Aist and Bayles, 1991d). In such a case, this tugging on mitochondria could contribute to the astral pulling force during anaphase B, especially if the mitochondria were anchored to cytoskeletal structures, such as F-actin, intermediate filaments, or cytoplasmic MTs.

Ultrastructural analyses (Aist and Bayles, 1991a; Jensen et al., 1991) of asters in F. solani f. sp. pisi provided a wealth of new structural information. The MTs of these asters are of three types: attached to the SPB, not attached to anything, and not attached to the SPB but likely cross-bridged to other MTs that are attached. Many of the astral MTs were shown to be cross-bridged to other astral MTs. The deployment of MTs in the mitotic asters gave the impression that the MTs were exhibiting dynamic instability and were in a state of tension which pulled them from their moorings at the cytoplasmic surface of the SPB. The two asters of an anaphase B nucleus may be of different sizes at any given moment, which could explain how the astral pulling force could fluctuate and produce the oscillations of the entire mitotic apparatus that are characteristic of this stage (Aist and Bayles, 1988). Putative cross-bridges between astral MTs were reported to occur also in the mitotic apparatus of a basidiomycete (O'Donnell, 1994).

Finally, the distal tips of a few of the astral MTs were shown, as early as 1981, to be associated with the plasma membrane via a localized, fibrillogranular, flocculent material located in the cell cortex subjacent to the plasma membrane at the point of astral MT termination (Fig. 7; Aist and Bayles, 1991a; Aist and Berns, 1981). Other astral MTs were associated laterally with linear elements in the astral regions that were of the diameter and appearance of intermediate filaments (Aist and Bayles, 1991a). Either or both of these astral MT associations could theoretically contribute to the astral pulling force. Many recent studies of mitotic asters in fungi have dealt with the relationships of mitotic asters with the plasma membrane in yeasts (Goode et al., 2000; Heil-Chapdelaine et al., 1999) and a basidiomycete (O'Donnell, 1994).

Fig. 7. An electron micrograph showing the plus end of an astral microtubule (AMT) of an anaphase B nucleus of Fusarium solani f. sp. pisi embedded in a tuft of thin filaments (arrowheads) in the cell cortex, subjacent to the plasma membrane. GE, Golgi equivalent. Scale bar-0.1 μm.

 (from Aist and Bayles, 1991a).

The inference that mitotic spindles in filamentous ascomycetes develop within them an outwardly directed force (herein called the "spindle pushing force") that pushes against the SPBs and aids in their separation (i.e., spindle elongation) during anaphase was based on the evidence of a few illustrations of bent spindles, several of which were published about a century ago (Aist and Morris, 1999). Recently, precise measurements of spindle length during episodes of spindle bending in living cells of F. solani f. sp. pisi (Aist and Bayles, 1991c) demonstrated that spindle bending is fairly common but fleeting, and that anaphase spindles bend because of pushing forces that are developed within them. This finding was concordant and virtually contemporary with the earliest discoveries that MT-associated motor proteins in the spindles of other fungi are involved in the production of force for spindle elongation (Enos and Morris, 1990; Hagan and Yanagida, 1990; Meluh and Rose, 1990).

During anaphase B in F. solani f. sp. pisi, the shortest MTs were selectively depolymerized without evidence of any MT growth taking place in the spindle (Aist and Bayles, 1991b). Coupled with the extensive lengthening of the spindle at this stage, this depolymerization led to a visible reduction in the diameter of the spindle during anaphase B, which was reflected in a much smaller number of spindle MT profiles in an average cross section (Jensen et al., 1991). No continuous, pole-to-pole MTs were found in the anaphase B spindle, but the MT fragments comprising the spindle were heavily cross-bridged (Jensen et al., 1991), apparently preserving the integrity of the spindle as well as its force-generating capability. The overall results of detailed, fine-structural analyses of MTs making up the spindle of F. solani f. sp. pisi (Fig. 6; Aist and Bayles, 1991b; Jensen et al., 1991)—especially the lack of a central, overlap zone of antiparallel MTs—led to the development of a "telescoping" model for spindle elongation in which the spindle would be elongated primarily by the astral pulling force much like one would extend a telescope. According to this model, the MT fragments comprising the spindle would be slid passively past each other, giving rise to both spindle elongation and a narrowing of the spindle during anaphase B, as was observed (Aist and Bayles, 1991b).

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Mitosis and Cytokinesis

In Cell Biology (Third Edition), 2017

Mitotic Spindle Dynamics and Chromosome Movement During Anaphase

Anaphase is dominated by the orderly movement of sister chromatids to opposite spindle poles brought about by the combined action of motor proteins and changes in microtubule length. There are two components to anaphase chromosome movements ( Fig. 44.15). Anaphase A, the movement of the sister chromatids to the spindle poles, requires a shortening of the kinetochore fibers. During anaphase B, the spindle elongates, pushing the spindle poles apart. The poles separate partially because of interactions between the antiparallel interpolar microtubules of the central spindle and partially because of intrinsic motility of the asters. Most cells use both components of anaphase, but one component may predominate in relation to the other.

Microtubule disassembly on its own can move chromosomes (see Fig. 37.8). Energy for this movement comes from hydrolysis of GTP bound to assembled tubulin, which is stored in the conformation of the lattice of tubulin subunits. Microtubule protofilaments are straight when growing, but after GTP hydrolysis protofilaments are curved, so they peel back from the ends of shrinking microtubules (see Fig. 34.6). Several kinesin "motors" influence the dynamic instability of the spindle microtubules. Members of the kinesin-13 class, which encircle microtubules near kinetochores and at spindle poles, use adenosine triphosphate (ATP) hydrolysis to remove tubulin dimers and promote microtubule disassembly rather than movement.

Kinetochores are remarkable in their ability to hold onto disassembling microtubules. In straight (growing) microtubules, the Ndc80 complex is mostly responsible for microtubule binding. It binds to the interface between α and β tubulin subunits. This interface bends in curved (shrinking) microtubules, so Ndc80 cannot bind. This could allow it to redistribute onto straight sections of the lattice and thereby move away from the curved protofilaments at the disassembling end. In metazoans the Ska complex in the outer kinetochore binds α and β tubulin subunits away from the interface, so it can bind to curved (disassembling) protofilaments. At yeast kinetochores the Dam1 ring (green in Fig. 8.21) couples the kinetochore to disassembling microtubules.

Anaphase A chromosome movement involves a combination of microtubule shortening and translocation of the microtubule lattice that result from flux of tubulin subunits (Fig. 44.14). The contributions of the two mechanisms vary among different cell types. When living vertebrate cells are injected with fluorescently labeled tubulin subunits, the spindle becomes fluorescent (Fig. 44.17). If a laser is used to bleach a narrow zone in the fluorescent tubulin across the spindle between the chromosomes and the pole early in anaphase, the chromosomes approach the bleached zone much faster than the bleached zone approaches the spindle pole. This shows that the chromosomes "eat" their way along the kinetochore microtubules toward the pole. In these cells, subunit flux accounts for only 20% to 30% of chromosome movement during anaphase A, and this flux is dispensable for chromosome movement. In Drosophila embryos, in which subunit flux accounts for approximately 90% of anaphase A chromosome movement, the chromosomes catch up with a marked region of the kinetochore fiber slowly, if at all.

Anaphase B appears to be triggered at least in part by the inactivation of the minus-end–directed kinesin-14 motors, so that all the net motor force favors spindle elongation. Four factors contribute to overall lengthening of the spindle: release of sister chromatid cohesion, sliding apart of the interdigitated half-spindles, microtubule growth, and intrinsic motility of the poles themselves (Fig. 44.7). During the latter stages of anaphase B, the spindle poles, with their attached kinetochore microtubules, appear to move away from the interpolar microtubules as the spindle lengthens. This movement of the poles involves interaction of the astral microtubules with cytoplasmic dynein molecules anchored at the cell cortex.

Anaphase B spindle elongation is accompanied by reorganization of the interpolar microtubules into a highly organized central spindle between the separating chromatids (Fig. 44.15). Within the central spindle, an amorphous dense material called stem body matrix stabilizes bundles of antiparallel microtubules and holds together the two interdigitated half-spindles. Proteins concentrated in the central spindle help regulate cytokinesis. One key factor, PRC1 (protein regulated in cytokinesis 1), is inactive when phosphorylated by Cdk kinase and functions only during anaphase when Cdk activity declines and phosphatases remove the phosphate groups placed on target proteins by Cdks and other mitotic kinases. PRC1 directs the binding of several kinesins to the central spindle. The kinesin KIF4A targets Aurora B kinase to a particular domain of the central spindle, where phosphorylation of key substrates then regulates spindle elongation and cytokinesis.

How can protein kinases such as Aurora B continue to function during anaphase while protein phosphatases are removing phosphate groups placed there by Cdks and, indeed, Aurora B during early mitosis? One answer is that the phosphatase activity is highly localized, controlled by specific targeting subunits. Cdk phosphorylation can inhibit targeting subunits such as the exotically named Repo-Man (recruits PP1 onto mitotic chromatin at anaphase) from binding protein phosphatase 1 or localizing to targets, such as chromatin in early mitosis. When Cdk activity drops, Repo-Man (and other similar targeting subunits) is dephosphorylated, and now targets PP1 to chromatin, where it removes phosphates placed there by Aurora B in the CPC. As long as phosphatases are not specifically targeted to the cleavage furrow, Aurora B can continue to control events there during mitotic exit by phosphorylating key target proteins required for cytokinesis.

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Molecular Motors and Motility

S. Dumont , T.J. Mitchison , in Comprehensive Biophysics, 2012

Glossary

Anaphase A

Stage of mitosis when the chromosomes separate and move towards the spindle poles.

Anaphase B

Stage of mitosis when the spindle poles separate.

Biased diffusion

Diffusion of a particle whose net motion is strongly biased in one direction by an energy source.

Centromere

Functional center of a chromosome where the sister chromatids are held and where the kinetochore is built.

Centrosome

Organelle serving as the main microtubule organizing center in metazoans.

Chromokinesin

Kinesin motor located on chromosome arms.

Kinetochore

Macromolecular assembly built on the centromere that mediates the attachment of chromosomes to spindle microtubules.

Metaphase

Stage of mitosis when chromosomes are positioned at the spindle equator in a brief steady state.

Polar ejection force

A microtubule-dependent force that pushes chromosomes away from spindle poles.

Poleward flux

Continuous spindle microtubule sliding towards spindle poles.

Power stroke

Stroke of a motor (conformational change) which generates mechanical force from chemical potential.

Prometaphase

Stage of mitosis when the spindle begins to form and microtubules begin to attach to kinetochores.

Prophase

Stage of mitosis when the chromosomes start to condense and the nucleus starts to break down.

Speckle imaging

Under-labeling of periodic cellular components (e.g., filaments) such that, instead of appearing continuous, they appear as discrete speckles that can reveal component dynamics.

Spindle

Cellular assembly based on a bipolar array of microtubules that segregates chromosomes during eukaryotic cell division.

Spindle matrix

A controversial microtubule-independent network proposed to provide a structural scaffold to the spindle.

Telophase

Stage of mitosis when the spindle disappears and the two nuclei form.

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Electron Microscopy of Model Systems

Thomas H. GiddingsJr., ... Mark Winey , in Methods in Cell Biology, 2010

I Introduction

Centrioles primarily serve two functions in cells. They comprise the core of centrosomes and mitotic spindle poles, and they act as basal bodies (BB) to template the formation of cilia. Regardless of their cytological role or cell type, the basic structure, consisting of a cylinder of microtubules arranged in 9-fold symmetry, is highly conserved ( Beisson and Wright, 2003). A number of ciliated or flagellated cell types have been developed as model systems to investigate centriole and BB structure, function and mechanism of assembly (e.g. Marshall and Rosenbaum, 2000; O'Toole et al., 2007; Pearson and Winey, 2009). In particular, Tetrahymena thermophila are single-cell, motile, ciliated protists containing approximately 750 basal bodies per cell. Here, we present methods to analyze the structure of assembling and mature T. thermophila BBs at high resolution and in three dimensions. These methods can be combined with molecular techniques to investigate the structure and function of components by gene disruption and localization of tagged proteins.

Approximately 600 basal bodies are aligned in rows in the cortical cytoplasm of T. thermophila to form the cilia responsible for motility (Fig. 1). About 150 more are tightly packed in the oral apparatus, a cavity involved in nutrient uptake. In Tetrahymena and other ciliates, cortical basal bodies anchor a complex network of cytoskeletal elements arrayed in a reiterated pattern. Each basal body nucleates the microtubules (MTs) of the ciliary axoneme and is associated with at least two other MT arrays, the transverse MTs and the post-ciliary MTs (Allen, 1969; Dippell, 1968; Iftode and Fleury-Aubusson, 2003). Mature Tetrahymena BBs are approximately 200   nm in diameter and 600   nm in length (Fig. 2). The proximal end is defined as the base, located in the cytoplasm facing the cell center, and the distal end is linked with the cilium (Allen, 1969). The wall of the basal body is comprised of nine MT triplets arranged in a cylinder (Fig. 3). Near the proximal end, the triplets are noticeably angled toward the center. The innermost MT is designated the "a" tubule, the middle MT as "b" and the outermost as the "c" tubule (Fig. 3B). The a and b MTs of the basal body triplets are continuous with the outer doublet MTs of the cilia. A cartwheel-shaped structure occupies the center of the BB cylinder at the proximal end (Fig. 2). A long filamentous structure, the kinetodesmal (KD) fiber, attaches laterally to the proximal end of the BB (Fig. 3). The formation of new basal bodies begins near the site of attachment of the KD fiber (Fig. 3). An electron dense core occupies the central BB lumen extending from the cartwheel to the transition zone (Fig. 2). Near the cell surface, the transition zone (TZ) marks the distal end of the BB (Fig. 2). The outermost MT of each BB triplet terminates at the TZ. Distal to the TZ is the axoneme of the cilium. The central pair of MTs of the axoneme is anchored in an electron density known as the axosome on the distal side of the TZ.

Fig. 1. Immuno-fluorescence micrograph of a Tetrahymena cell labeled with an antibody to centrin, a pan specific marker for BBs (Stemm-Wolf et al., 2005). Cortical rows of BBs run the length of the cell. The oral apparatus is located near the anterior of the cell (top). Scale bar   =   10   µm.

Fig. 2. Electron micrograph of a longitudinal section through a cortical row BB and attached cilium from a HPF/FS-fixed Tetrahymena cell. The hub of the cartwheel (CW) is visible at the proximal end of the BB. The core density (CD) extends from the top of the CW nearly to the transition zone (TZ). On the distal surface of the TZ, an electron-dense area known as the axosome (Axo) anchors one of the central pair (CP) MTs of the cilium's axoneme. Outer doublet (OD) MTs of the axoneme are continuous with the triplet MTs that form the BB cylinder. Some of the structures associated with the proximal end of the BBs are visible in this image, including one of the transverse microtubules (TMT) and the collar (Col). The cell anterior is to the left in this view; HPF/FS thin section (Epon). Scale bar   =   100   nm.

Fig. 3. Cortical row BBs in cross section. (A) Three BBs in a cortical row (left side) and two cilia (right side) are seen in cross section. A band of post-ciliary MTs (PCT) lies adjacent to each BB along the side facing the posterior of the cell. The KD fiber (KD) attaches to the anterior-facing side of the BB at the proximal end. The core density (CD) is visible in the lower two BBs. Part of the axosome and a single central pair MT (arrow) is visible in the upper BB. (B) Higher magnification view of a BB in cross section showing a KD fiber, post-ciliary MTs, some of the collar (Col) and the site of nascent BB assembly (bracket). The a, b, and c MTs of one of the BB triplets are identified; HPF/FS thin sections (Epon). Scale bars   =   100   nm.

In addition to making it possible to sample many BBs in a few micrographs, the large number of BBs and repetitive nature of the ciliary row organization make it feasible to find transient structures such as early stages of assembly. The position of new BBs relative to the mother BBs is predictable; they are always assembled on the anterior side at the proximal end. Synchronized cultures where most of the cells (>95%) have a high frequency of assembling BBs enable straightforward identification of basal body assembly intermediates. The abundance of basal bodies in Tetrahymena makes this organism an excellent model system for ultrastructural analysis of basal body and cilia biogenesis. Often multiple BBs can be viewed in cross section at sequential levels along the long axis of the basal body in a single micrograph (Fig. 4). A number of molecular techniques have been developed in recent years that allow the use of Tetrahymena as a versatile model system for experimental cell biology (reviewed by Turkewitz et al., 2002). The complete Tetrahymena genome has been sequenced (Eisen et al., 2006) and genetic tools have been developed to construct strains with epitope-tagged gene products, inducible gene expression, and gene deletions (Bruns and Cassidy-Hanley, 2000a,b; Frankel, 2000; Gaertig and Kapler, 2000; Hai et al., 2000; Malone et al., 2008; Pearson and Winey, 2009; Pearson et al., 2009b; Stemm-Wolf et al., 2005; Yu and Gorovsky, 2000). Kilburn et al. (2007) have characterized a Tetrahymena BB proteome. In addition to the genetic tools, basal body duplication can be temporarily suppressed by cell cycle arrest or synchronized basal body amplification can be induced to maximize the frequency of BB duplication at a known time point (Pearson et al., 2009b). Experiments related to basal body assembly, maintenance and turnover are now possible (Pearson and Winey, 2009).

Fig. 4. Thin section through an oral apparatus showing BBs cross sectioned at different levels along their long axis. The basal bodies toward the right side of the image were sectioned through the cartwheel structure located at the proximal end. HPF/FS. Scale bar   =   100   nm.

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