YEASTBOOK CELL STRUCTURE & TRAFFICKING Mitotic Spindle Form and Function Mark Winey* and Kerry Bloom†,1 *Molecular Cell and Developmental Biology, University of Colorado, Boulder, Colorado 80309 and †Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280 ABSTRACT The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine. TABLE OF CONTENTS Abstract 1197 Introduction 1198 Spindle Structure 1198 The Parts List 1199 Spindle pole bodies 1199 Microtubules 1203 Microtubule motor proteins 1204 Microtubule-associated proteins 1205 DNA, cohesion, and condensin springs in the spindle 1207 The kinetochore 1208 Building the Spindle 1209 Spindle pole duplication and separation 1209 Regulation of SPB duplication 1212 Spindle Dynamics 1213 Regulation of microtubule dynamics, kinetochore and interpolar microtubules 1213 Regulation of spindle length and stability 1214 Spindle Orientation and Translocation 1215 Continued Copyright © 2012 by the Genetics Society of America doi: 10.1534/genetics.111.128710 Manuscript received March 15, 2011; accepted for publication September 6, 2011 1Corresponding author: CB #3280, Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280. E-mail: [email protected] Genetics, Vol. 190, 1197–1224 April 2012 1197 CONTENTS, continued Regulation of spindle orientation and translocation 1216 Spindle Disassembly: Mitotic Exit and Preparation for the Next Cycle 1217 Prospective 1217 N the budding yeast Saccharomyces cerevisiae, the mitotic is constant for 15–20 min. At this stage the spindle appears Ispindle is exemplified by its simplicity and elegance. Mi- fairly rigid and can rotate up to 90° obliquely to the mother/ crotubules (MTs) are nucleated by the highly organized ar- bud axis. Anaphase onset is characterized by rapid linear ray of proteins inserted in a fenestra in the nuclear envelope elongation of the spindle with a velocity of 1 mm/min to known as the spindle pole body (SPB, analogous to the a length of 6 mm. These rates are consistent with earlier centrosome in larger eukaryotes). The SPB organizes two measurements taken from low-level DAPI-stained cells classes of nuclear spindle microtubules: kinetochore (KT) (Palmer et al. 1989). In anaphase the kinetochore microtu- and interpolar microtubules. One kinetochore microtubule bules shorten from their plus-end (anaphase A). As spindle attaches to a single centromere on each chromosome, while elongation ensues (anaphase B), the number of interpolar approximately four interpolar microtubules emanate from microtubules decreases to one or two interpolar microtubules each pole and interdigitate with interpolar microtubules from each pole prior to spindle disassembly. from the opposite spindle to provide stability to the bipolar The lack of detectable kinetochore structures required spindle. On the cytoplasmic face, two to three microtubules that kinetochore microtubules be detected by various compu- extend from the SPB toward the cell cortex. Processes re- tational techniques. Two key findings about the kinetochore quiring microtubule function are limited to spindles in mi- microtubules came of these analyses. First, the plus ends of tosis and spindle orientation and nuclear positioning in the the kinetochore microtubules from opposing SPBs were never cytoplasm. Microtubule function is regulated in large part found to be near each other as one might expect at metaphase via products of the 6 kinesin genes and the 1 cytoplasmic when the paired sister chromatids achieve bipolar spindle dynein gene. A single bipolar kinesin (Cin8, class Kin-5), attachment. What was observed was a gap of 250 nm be- together with a depolymerase (Kip3, class Kin-8) or mi- tween the ends of the kinetochore microtubules from each nus-end-directed kinesin (Kar3, class Kin -14), can support SPB, leading to the suggestion that the sister centromeres spindle function and cell viability. The remarkable feature were separated from each other at metaphase in yeast (Winey fi that both budding and ssion yeast cells can survive with et al. 1995). This proposal was confirmed by fluorescent mi- microtubules and genes for just a few motor proteins pro- croscopy studies of live cells with marked tubulin (Tub-GFP, vides an unparalleled system to dissect microtubule and Figure 1) centromeric DNA or kinetochores (Goshima and motor function within the spindle machine. Yanagida 2000; Tanaka et al. 2000; He et al. 2001; Pearson et al. 2001). The position of lacO arrays relative to the cen- tromere is critical to visualizing separated sister centromeres. Spindle Structure The most proximal centromere marked DNA spots [within 1 Three-dimensional ultrastructural analysis of yeast mitotic kilobase (kb) pairs from the centromere] are spatially sepa- spindle microtubules has been accomplished by reconstruc- rated for the duration of mitosis (Goshima and Yanagida tion from serial thin sections of cells (Winey et al. 1995) and 2001; Pearson et al. 2001). Second, the appearance of clusters by electron tomography of thick sections of cells (O’Toole of sister kinetochore proteins provide evidence for metaphase et al. 1999) (Figure 1). The work confirmed that kineto- in budding yeast (Pearson et al. 2001). Third, the electron chores are attached by a single microtubule as suggested tomography confirmed that anaphase A (kinetochores moving in early high-voltage electron microscopy (EM) studies of toward the poles) occurred in budding yeast (O’Toole et al. isolated yeast spindles (Peterson and Ris 1976). Reconstruc- 1999). Chromosome movement in anaphase proceeds at a rate tion of yeast mitotic spindle microtubules from serial thin of 1 mm/min. As a point of reference, if you read down this sections or electron tomograms revealed that the spindle is page at the rate a yeast chromosome moves, it would take a highly stereotypic microtubule array in these cells. In you 150 days to get to the foot of the page. Anaphase B metaphase, it is 1.4–1.5 mm long, and haploid cells contain (increasing pole-to-pole distance) is so significant in yeast 20 microtubules from each SPB. These microtubules can (the 1.5-mm metaphase spindle will lengthen to nearly be divided into shorter microtubules that do not interact 10 mm) that this movement alone may be sufficient to sepa- with antiparallel microtubules from the other SPB, suggest- rate sister chromatids. However, the reconstructions revealed ing that they are kinetochore microtubules, particularly that the kinetochore MTs of 0.5 mmlongatmetaphase since there are very nearly 16 per haploid SPB (Winey (Figure 1) are shorter in anaphase cells. In fact, they shrink et al. 1995). Once formed, the 1.5- 2.0-mm spindle length all the way to 30–50 nm in late anaphase. These remnants 1198 M. Winey and K. Bloom tween these microtubules that could be various motors or microtubule-associated proteins (MAPs), but those molecu- lar identifications have not been made (Winey et al. 1995). Finally, the long microtubules of the anaphase B central spindle seem to persist into G1 after the spindle is severed during karyokinesis. The fact that the budding yeast SPB is embedded in the nuclear envelope throughout the entire life cycle of the organism is consistent with the observation that microtubules are continually present in the nucleus. The G1 nuclear microtubule array have a median length of 150 nm (O’Toole et al. 1999). The Parts List Figure 1 Yeast mitotic spindle structure. Sixteen kinetochore microtubules and four interpolar microtubules emanate from each spindle pole in a hap- Spindle pole bodies loid cell: 40 MTs/1.5 mm spindle. (Left) The yeast mitotic spindle as seen in the electron microscope (EM) using thin sections of high-pressure frozen The S. cerevisiae SPB was first observed in the electron mi- and freeze-substituted cells (e.g.,Wineyet al. 1995). The budded cell con- croscope by Robinow and Marak (1966) (Figure 2). As the tains two SPBs (black arrows) that form a bipolar spindle (microtubules are sole microtubule-organizing center (MTOC) in these cells the filaments between the SPBs in the nucleus). The nuclear envelope (white carets) extends through the bud neck, a typical configuration at this that have a closed mitosis (the nuclear envelope stays intact point in the cell cycle. (Right) The yeast mitotic spindle as seen in the light throughout the cell cycle), the SPB must have access to the microscope using a-tubulin fused to green fluorescent protein (Tub-GFP, nucleoplasm to form the microtubules of the mitotic spindle shown in Figure as white protein on black background). The outline of the and to the cytoplasm to form the astral microtubules that cell is illustrated by the outline in white. The spindle can be seen at the bud will position the nucleus.