How does an interphase nucleus find the cell center? The cylindrically shaped cells of fission yeast have a centrally placed nucleus and divide by fission at the cell center. A direct way to study how the cell positions its nucleus in the center involves mechanical perturbation of the nuclear position. We have developed a technique to move the nucleus within the cell using optical tweezers. These optical manipulations offer several advantages over the methods previously used to displace cell organelles, such as centrifugation: organelles can be displaced selectively; the manipulation can be performed exactly at a chosen time in the cell cycle; and, displacements as small as a few hundred nanometers can be detected because the image of the cell after the manipulation can be compared directly to the image before the manipulation. Our experiments show that when we displace the nucleus away from the cell center using optical tweezers, and then switch off the tweezers, the nucleus returns to the cell center. The force for the return of the nucleus is provided by microtubules, which push against the cell ends and thereby move the nucleus towards the middle of the cell. We are currently investigating the regulatory mechanisms of these pushing forces.
How does a cell determine its division site? We are interested in the spatio-temporal coordination of nuclear events (chromosome segregation) and cortical activities (cytokinesis). Is the spatial cue for the division site provided by the position of the nucleus? If the division site is established by a signal from the nucleus at a defined period of the cell cycle, then displacing the nucleus at an earlier time would result in a corresponding displacement of the division plane, whereas a later displacement would not affect the normal position of the division plane at the cell center. Our experiments, in which we displace the nucleus by optical tweezers, suggest that the division plane is indeed specified by the position of the nucleus. Moreover, the cell makes the decision where to divide at the very beginning of mitosis. Why so early, when a number of other cell types make that decision by the end of mitosis? The answer probably lies in the fact that the fission yeast nucleus is centered by microtubules during interphase but not in mitosis. Thus the establishment of the division plane at the beginning of mitosis may be an optimal mechanism for accurate division in these cells.
Displacement of the cell nucleus (red: before; green: after) using optical tweezers. Maghelli and Tolic-Nørrelykke, J. Biophoton. 2008
Mitotic Spindle Positioning
In all eukaryotic cells, spindle position with respect to the cleavage plane is important for a successful segregation of whole chromosome sets into daughter cells. In some cells, spindle axis determines the cleavage plane, thus also the size and fate of daughter cells. When and how does the spindle get aligned with the cell axis? There seem to be several complementary mechanisms by which the spindle becomes properly aligned. Using confocal microscopy and high-precision tracking of spindle poles, we have identified two mechanisms of spindle alignment in fission yeast. Both mechanisms are at work during anaphase. One is based on cell geometry: the fixed cylindrical shape of the cell forces an elongating spindle to align with the main cell axis. In the other mechanism, astral microtubules push on the spindle poles against the cell edge, which helps to center the poles and thus to align the spindle. We have recently discovered a new mechanism of spindle alignment, which is active in the beginning of mitosis. The spindle is remarkably well aligned with the cell longitudinal axis at the onset of mitosis, by growing along the axis of the adjacent interphase MT. Misalignment of nascent spindles can give rise to anucleate cells when spindle elongation is impaired.
(A) Spindle elongation and alignment. (B) Model of alignment of nascent spindles by interphase microtubules. Vogel et al., Curr. Biol. 2007.
As the spindles grow, they rotate and lose their initial alignment. Is there a mechanism that constrains spindle rotation, preventing them to flip around or become strongly mislaigned? We show that the association of mitochondria with the spindle poles reduces spindle rotation. In wild type, spindles with associated mitochondria do not rotate as much as free spindles.
(A) Electron micrograph of a wild-type S. pombe cell in mid-mitosis. A mitochondrion (Mi) is found in close proximity to each spindle pole body (SPB). (B) A typical wild-type cell shows persistent association of mitochondria with the spindle poles throughout mitosis, and only modest rotation of the spindle. (C) A rare wild-type cell, where the mitochondrial association with spindle poles was lost, shows extensive spindle rotation.
In a mutant of the centrosomin-related protein Mto1p, mitochondria were less associated with the spindles, which rotated more than in wild type. There seem to be a symbiotic relationship between mitochondria and the mitotic spindle, where close association between the two organelles facilitates the positioning of both: while the spindle helps to segregate mitochondria equally among the nascent daughter cells (Yaffe et al., 2003), mitochondria decrease spindle rotation and thus promote spindle alignment.
Mitochondria decrease spindle rotation and thus promote spindle alignment. Left: The spindle (green) is initially aligned with the cell axis. Mitochondria (red), which are associated with the spindle poles, decrease the rotation of the spindle and thus help the spindle to remain aligned with the cell axis. Right: Spindles lacking mitochondrial association rotate and lose their initial alignment. The resulting spindle misalignment may be fatal, leading to chromosome mis-segregation if spindle elongation is impaired.
Meiotic Nuclear Oscillations
What drives nuclear oscillations in meiotic prophase? In the meiotic prophase of fission yeast, the nucleus and the chromosomes oscillate from one end of the cell to the other (Chikashige et al., 1994). These oscillations follow the oscillatory movement of the spindle pole body (SPB, yeast centrosome). The force for the SPB movement is generated by the minus-end directed motor dynein (Yamamoto et al., 1999). It has been proposed that the oscillatory movement of the nucleus helps the alignment and pairing of the homologous chromosomes, by stretching and dragging the chromosomes. Indeed, mutants and conditions where the nucleus does not oscillate result in reduced recombination rates and spore viability (Ding et al., 2004; Yamamoto et al., 1999). These oscillations are not just a peculiarity of fission yeast, because similar chromosome movements have been observed in meiotic prophase in a variety of model organisms, from budding yeast to mouse (Parvinen and Soderstrom, 1976; Trelles-Sticken et al., 2005), and the role of these movements in chromosome pairing and recombination has been demonstrated (Scherthan et al., 2007). Even though meiotic nuclear oscillations have an important and most likely conserved biological role, the underlying physical mechanism is unknown.
Oscillation of the spindle pole body.
Aging
Fission yeast divides symmetrically. However, in spite of the morphological symmetry, daughter cells do not inherit equal sets of cellular components. The key questions are: which components segregate differentially, and whether asymmetric inheritance affects function and aging of the daughter cells. This work could establish fission yeast as a model system for cellular aging. If, however, the experiments show no signs of replicative aging, the challenge will be to understand what makes these cells immortal.
A spore gives rise to a colony of cells.
A pedigree tree of a microcolony of fission yeast cells. The length of each line is proportional to the time between two cell divisions.
