A kinesin mutant with an atypical bipolar spindle undergoes normal mitosis

Abstract

Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles.

Figure 1

ATK1 expression. Using RT-PCR with primers specific to ATK1, a 0.5-kb band was amplified in wild-type (WT) root tips, but not in homozygous atk1-1 roots (top lanes). As a control for RNA loading, RT-PCR amplification of rRNA in the two samples was adjusted to give equal intensity of a 0.4-kb band, when run on a 1% agarose gel (bottom lanes).

Figure 2

Root mitosis of wild-type and mutant plants. Immunofluorescence was performed using antitubulin antibodies in dividing root cells, in both wild-type (A, C, E, and G) and homozygous mutant (I, K, M, and O) cells; the adjacent image shows the same cell stained for chromosomes (B, D, F, and H for wild-type; J, L, N, and P for mutant). In wild-type (A) and mutant cells (I), the preprophase microtubule band (arrows) and perinuclear microtubule array are present in prophase (nucleus = N). A wild-type metaphase cell (C) has a well-defined equatorial zone of microtubule clearing (arrow) and a distinct bipolar spindle with chromosomes aligned linearly along the metaphase plate (D). A mutant metaphase cell lacks an equatorial zone of microtubule clearing (K) and chromosomes do not align linearly along the metaphase plate (L); as a result there is an indistinct bipolar spindle axis. Wild-type and mutant cells have a normal anaphase spindle (E and M, respectively) and chromosomes segregate normally (F and N, respectively). Wild-type and mutant cells in telophase (G and O, respectively) show a normal microtubule-based phragmoplast and the start of chromosome decondensation (H and P, respectively). Scale bar, 10 μm.

Figure 3

Mitotic indices of wild-type and mutant cells. The percentage of cells in preprophase, prophase, anaphase, and telophase in wild-type and mutant cells are not statistically different (p > 0.05 for all). However, mutant cells have significantly more cells in prometaphase/metaphase as compared with wild-type cells (p < 0.05). Error bars represent SE. The Student's t test was used to calculate p values.

Figure 4

Quantitative analysis of prometaphase/metaphase spindles. Spindles were classified into three categories: early prometaphase, late prometaphase, and metaphase (top). The results were quantitated in a histogram (bottom). Early prometaphase spindles typically lack bipolar character and an equatorial zone of microtubule clearing. Late prometaphase spindles exhibit more bipolarity than early prometaphase spindles, although an equatorial zone of microtubule clearing is still not robust. Metaphase spindles are bipolar and have a distinct equatorial zone of microtubule clearing (top panel). Mutant cells have significantly less early prometaphase and late prometaphase spindles and more metaphase spindles (bottom panel; p < 0.05). Error bars, SE. The Student's t test was used to calculate p values.

Figure 5

In planta temporal analysis of mitosis. For each genotype, time intervals for various stages of mitosis were quantified. The amount of time it took to proceed from PPB disappearance to the onset of anaphase was greater in ATK1/atk1-1 plants (mean = 608.7 ± 28.7 s) compared with ATK1/ATK1 plants (mean = 455.3 ± 64.7 s). However, atk1-1/atk1-1 plants took significantly longer (mean = 825.6 ± 106 s; p < 0.05), compared with the other genotypes. For all genotypes, the time it took for anaphase and from the completion of anaphase to the disappearance of the phragmoplast was not statistically different (p > 0.05). Error bars, SE. The Student's t test was used to calculate p values.

Figure 6

In planta spatial analysis of microtubules in mitosis. Shown here is a time course of mitosis. At t = 0 s, all genotypes have a normal perinuclear array (N = nucleus) and a PPB (arrows). In ATK1/ATK1 cells at t = 30 s and t = 60 s, microtubules increase at the incipient spindle poles, and by t = 150 s, microtubules radiate from the spindle poles. The beginning of a bipolar spindle is seen at t = 150 s, and by t = 450 s, anaphase typically occurs. In contrast, at t = 30 s and t = 60 s in both ATK1/atk1-1 and atk1-1/atk1-1 genotypes, microtubule accumulation is not specific to the spindle pole regions and a high density of microtubules are observed in the former nuclear region. At t = 150 s in ATK1/atk1-1 cells, a disorganized spindle is seen, which resolves into a mature spindle at t = 570 s, and anaphase typically occurs by t = 630 s. In atk1-1/atk1-1 cells at t = 150 s and at t = 270 s, microtubules continue to accumulate in the former nuclear region, and a mature spindle is not seen until t = 870 s. Anaphase typically occurs by t = 990 s. Scale bar, 10 μm.

Figure 7

Spindle pole formation in wild-type and mutant plants. For each genotype a time course was done to look at the early events of spindle pole formation, starting at preprophase and ending at early prometaphase. In ATK1/ATK1 plants at t = 20 s, the perinuclear microtubule array dims in the region proximal to the PPB, and a clearing is observed at t = 40 s (boxed region). Furthermore, there is a concomitant increase in microtubules at the spindle poles (sp), and by t = 60 s, the microtubules emanating from the poles have extended into the clearing zone along the perinuclear array. In the mutant genotypes at t = 40 s, microtubule clearing in the region of the perinuclear array that is proximal to the PPB is not seen (boxed region) and microtubule localization is not increasing at the spindle poles (sp), but is in other areas along the perinuclear array. This continues at t = 60 s, though microtubules also appear in the nucleus. Scale bar, 10 μm.