To set the stage for a discussion of centering mechanisms, we will first review microtubule organization during the first cell cycle in fertilized eggs of the clawed frog Xenopus laevis
. Egg diameter in this species is ~ 1200 μm, which is ~ two orders of magnitude larger in length than typical cells in animal tissues12
. Some amphibian have even larger eggs13
. Time is normalized to fertilization (defined as 0) and first cleavage (defined as 1)14
. Typical absolute values for the 0–1 interval is ~90 min at 23°C. The lower portion of a Xenopus
egg is packed with large yolk granules, creating a density asymmetry that makes the egg orient under gravity15
. The lowest part of the egg is called the vegetal pole, and the upper part the animal pole. In Xenopus
, the animal half of the egg is brown-black due dark pigment in the cortex, while the vegetal half is white. Because of the packed yolk in the vegetal half of the egg, the distribution of free cytoplasm is not spherical, but looks more like a flattened hemisphere (). In discussing models for centering, we will neglect the vertical dimension, and concentrate on a horizontal plane through this hemisphere of cytoplasm, as shown in . All the following picture and cartoons depict positioning and movement in this plane. Asters can also move somewhat in the vertical plane, and we assume they do so by mechanisms similar to movement in the horizontal plane.
Figure 1 Cartoon of a frog egg shortly after fertilization. The vegetal part (bottom) is heavily filled with yolk. The sperm enters randomly at the animal (top) part of the egg. The radial grow of the sperm aster leads to the movement of the centrosome towards (more ...)
Before fertilization, the egg is arrested in metaphase of meiosis II with a relatively small meiotic spindle attached to the cortex at the animal pole of the egg16
. Sophisticated mechanisms ensure that only one sperm enters the egg, in the animal hemisphere17
. Fertilization generates a wave of elevated Ca++ in the cytoplasm that triggers anaphase in the meiotic spindle followed by extrusion of half the maternal DNA into the second polar body. The sperm carries the male DNA and two centrioles, which form a microtubule organizing center that initiates outgrowth of a dense radial array of microtubules with their plus ends presumably oriented outwards18
. This structure is called the sperm aster ( and ). The sperm aster’s diameter increases at a rate we estimate from images of embryos fixed at different times as ~30 μm/min. By ~0.5 the sperm aster grows to the point that its plus ends come close to the cortex all around the circle defined by the plane in 5
. By this time, the centrosomes have moved towards the center of the cell ()19, 20
. The centering is not perfect; they tend to be closer to the site of sperm entry than the opposite side (), but it is clear they have moved a long way from where the sperm entered the egg, at least 300 μm in most cases. Below, we will discuss models for how this centering movement of the centrosomes might be driven.
Figure 2 Overview of microtubule organization during the first cell cycle in X. laevis. Top: Immunostaining against tubulin, bar = 500 μm. Arrows indicate positions of centrosomes. Time (t) is normalized to first cleavage. Bottom: cartoon of corresponding (more ...)
As soon a microtubules from the sperm aster reach the female nucleus, it starts to move towards the center of the aster, presumably pulled by dynein attached to the nuclear envelope21
. In this way, the male and female pronuclei meet close to the centrosomes.
As the first mitosis is initiated, both nuclear envelopes, and the sperm aster, disassemble, and a mitotic spindle assembles ()22
. In smaller cells, such as the C. elegans
egg, the mitotic spindle finds the center of the cell using long astral microtubules9
, but in the frog egg is clear that the sperm aster is responsible for moving both the centrosomes and the DNA to approximately the cell’s center, and the spindle then forms in that spot. The metaphase spindle probably could not center itself in the frog egg, because its astral microtubules are much shorter than the radius of the egg ()22, 23
At anaphase, the sister chromatids separate, and the astral microtubules of the spindle start to grow out rapidly; again we estimate an elongation rate of ~15μm/min based on fixed images. Anaphase chromosome movement presumably starts with a conventional, kinetochore-based anaphase-A. Anaphase-B movement in these large egg cells is atypical, presumably to allow a large segregation distance when spindles are small relative to the egg. The sister DNA masses move apart rapidly over a distance much larger than the metaphase spindle length, the reach a position ~half way between the center of the egg and its periphery (). This requires that the DNA masses move ~250 μm in ~25 min. Approximately half this movement occurs while the DNA is still condensed, and half after the nuclear envelope has reformed23
. The origin of the forces that drive and direct this large anaphase-telophase segregation movement are unclear. The centrosomes are positioned a few tens of microns ahead of the moving nuclei, and appear to be pulling them, but it is far from clear why the centrosomes move apart in a straight line that is parallel to the spindle axis. Because these movements can be viewed as asters moving towards the center of a volume of cytoplasm, we suspect they may be driven by the same forces that cause centering of the sperm aster, which we discuss below.
The paired asters, we here call telophase-asters, originate in the centrosomes of the anaphase mitotic spindle and not only move the sister nuclei apart but are also responsible for determining the cleavage plane. It is believed that the site of cleavage furrow ingression is specified by a line along the cortex, normal to the direction of chromosome segregation, where the two antiparallel arrays of microtubules from the pair of asters come together the cortex 24
Besides their role in cell division and chromosome separation, microtubules are also involved in determining the future dorso-ventral axis of the embryo. In this paper we would like to concentrate on microtubules involved in centering. Detailed descriptions of microtubules involved in setting up dorso-ventral axes are presented elsewhere25–27
An interesting aspect of the organization of both the sperm aster (), and the subsequent telophase asters () is that they appear hollow in tubulin immunofluorescence images28
, as if many of the microtubules in the periphery of the aster do not have their minus ends located near the centrosome. We do not think this hollow aster image is an artifact of fixation or stain penetration, because higher microtubules density close to the outline of the asters can not only be seen near the egg’s surface but also deep inside ( and ). Also, we think it physically impossible that all the microtubules with plus ends at the periphery of the aster could have minus ends close to the centrosome, because of physical packing constraints. If all microtubules were continuous from center to periphery, their density in a plane tangential to the aster would have to scale as 1/radius2
as the plane moved outwards from the center, and as 1/radius in a plane that cut through the center of the aster. Our immunofluorescence images are completely inconsistent with this relationship, since the asters get brighter towards the outside not dimmer. We presume a subset of the astral microtubules are nucleated at centrosomes and run continuously out to the periphery, since the centrosome stays in the center of the aster as the aster moves and expands, implying the centrosome is physically connected to the aster periphery. But we believe that the majority of microtubules in these asters must have a different origin. Perhaps they are nucleated from the sides of existing microtubules, pointing in the same direction, for example. Microtubules are nucleated in the absence of centrosomes in egg meiotic spindles, and in this case too it may be important that new microtubules point in the same direction as the majority of microtubules near them, to preserve the gradient of polarity in each half spindle29
. We suspect that both situations require a biochemical mechanism that nucleates new microtubules in the vicinity of old ones, and pointing in the same direction.
Figure 4 How do asters notice each other? A) Sperm asters in polyspermic embryo space each other apart creating microtubule-sparse regions between them. Reprint by Herlant and Brachet (1910), kind permission of Springer Link.37 B) Immunostaining of telophase in (more ...)