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Address correspondence to Lesilee Rose, Section of Molecular and Cellular Biology, One Shields Ave., University of California, Davis, Davis, CA 95616. Tel.: (530) 754-9884. Fax: (530) 752-3085. E-mail: lsrose{at}ucdavis.edu
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Abstract |
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Key Words: asymmetric division; polarity; spindle orientation; PAR proteins; LET-99
* Abbreviations used in this paper: DIC, differential interference contrast; RNAi, RNA interference.
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Introduction |
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The Caenorhabditis elegans embryo is an excellent system in which to study spindle orientation, as it displays both symmetric and asymmetric divisions in a virtually invariant pattern. The spindle in the one-cell embryo (P0) orients onto the longitudinal axis of the embryo, which is also the polarized anterior/posterior axis. Division is asymmetric, producing an anterior AB cell and a posterior P1 cell. At second cleavage, AB divides symmetrically with a transverse spindle, while the P1 spindle is oriented again on the anterior/posterior axis (Rose and Kemphues, 1998b; Bowerman and Shelton, 1999). Longitudinal spindle orientation in P0 and P1 results from a 90° rotation of the nuclearcentrosome complex during prophase, which does not occur in AB.
Nuclear rotation in asymmetrically dividing cells is under the control of polarity cues. Polarity in the C. elegans embryos is established in the one-cell embryo through the asymmetric distributions of several PAR proteins, which are conserved in many organisms (Ohno, 2001). PAR-3 and PAR-2 are present at the anterior and posterior cortex, respectively, of both P0 and P1; PAR-3 is also present uniformly at the cortex of AB (Etemad-Moghadam et al., 1995; Boyd et al., 1996). Previous studies showed that nuclear rotation occurs in par-3 and par-3;par-2 double mutants in both one- and two-cell embryos, but not in par-2 single mutants (Cheng et al., 1995). These results combined with immunolocalization studies led to the model that in wild-type embryos, neither PAR-3 nor PAR-2 is required for rotation. Rather, it was proposed that PAR-3 somehow inhibits rotation in AB and the role of PAR-2 is to restrict the localization of PAR-3 to the AB cell cortex and the anterior cortex of P1 (Cheng et al., 1995; Etemad-Moghadam et al., 1995). However, this model cannot explain how nuclear rotation occurs in par-3 embryos where there is no apparent cellular polarity.
The molecular mechanism of nuclear rotation and how it is regulated by PAR proteins remains to be elucidated. In both the P0 and P1 cells, a process called centration occurs where nuclei migrate from the posterior to the center of the cell. During this time, the nuclearcentrosome complex usually begins rotation, and rotation is completed before nuclear envelope breakdown. Nuclear rotation depends on the function of the microtubule motor dynein in P0 cells. In P1 cells, the dynein-associated dynactin complex accumulates at a site on the anterior cortex, coincident with the position of the midbody/cell division remnant at the cell contact between P1 and AB. It has been proposed that dynein present at this dynactin-enriched cortical site captures and shortens astral microtubules, generating a pulling force that causes rotation and an extended anterior movement of the nucleus away from the center of cells (Fig. 1 A; Hyman and White, 1987; Hyman, 1989; Waddle et al., 1994; Keating and White, 1998; Skop and White, 1998; Gönczy et al., 1999; Gönczy, 2002). We refer to this type of movement as directed rotation, because the rotation appears directed toward the cell contact region and the nucleus becomes closely juxtaposed to the membrane. The observation that directed rotation occurs in both cells of par-3 embryos has been interpreted as evidence for cortical site activity in both cells (Waddle et al., 1994; Keating and White, 1998). However, in wild-type embryos, PAR-3 is present on both the AB and P1 side of the cell contact region by the time of rotation, and thus it is not clear how PAR-3 could inhibit the accumulation of dynactin and/or its function only in AB. Thus, although the cortical site model explains P1 rotation, how this cortical site is regulated by PAR polarity is unknown. Also, no such specialized site has been identified in P0, and there is no movement of the nucleus past the center of the cell during rotation in P0.
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Thus, a clear framework for how the PAR proteins regulate nuclear rotation has not been produced based on the current data. This suggests that other factors that influence spindle orientation remain to be found. Previously, we showed that nuclear rotation in one-cell par-3 embryos is not equivalent to wild-type rotation, but rather is due to an extrinsic cue, the oblong shape of the embryo (Tsou et al., 2002). In this paper, we re-evaluate the mechanism of nuclear rotation in both wild-type and par-3 two-cell embryos. The results challenge the cortical site model and support the LET-99 band model. Furthermore, the data clarify the role of the PAR proteins and indicates that PAR-3 has two roles: (1) it is required for an intrinsically programmed mechanism of nuclear rotation in asymmetrically dividing cells; and (2) is also required to inhibit geometry-dependent rotation in nonpolarized cells.
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Results |
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Interestingly, although membrane invaginations were never observed in spherical P1 cells (n = 12), membrane invaginations were frequently observed in the irradiated AB cells (n = 7/12). As stated earlier, the irradiation severely slowed down the AB cell cycle and prevented normal spindle formation. Membrane invaginations in AB cells appeared as the cell contact region curved into the AB cell concomitant with mitosis in the spherical P1 cell. The invaginations were centered around the cell division remnant (identified from the position of the midbody at first cytokinesis; Fig. 2, PS). This observation shows that membrane invaginations are able to form in these embryos, indicating that UV irradiation did not damage this process. Examination of the P1 cell in embryos with AB membrane invaginations showed that the centrosomes and spindles of P1 cells did not move or point toward the invagination during nuclear rotation and spindle elongation (Fig. 2, PS). These results indicate that the formation of a membrane invagination is independent of nuclear rotation and asymmetric spindle elongation in spherical P1 cells. Furthermore, invaginations in untreated wild-type embryos appear in P1 (Hyman, 1989), which has a flat or curved-in cell contact region, and invaginations in irradiated embryos only appeared in the irradiated AB cells with curved-in cell contact regions. These observations suggest that the invaginations are cell shape-dependent.
These results indicate that both the anterior position of the nucleus and the corresponding membrane invagination during rotation in wild-type P1 cells can be explained as a consequence of cell geometry, rather than being causally linked to the mechanism of rotation. Further, all of the observations on spherical wild-type P1 cells described are inconsistent with the cortical site model because the free nuclear rotation observed in spherical P1 cells cannot be explained by this model, regardless of whether the cortical site is present or not. Rather, these results indicate that nuclear rotation in P1 cells is controlled by mechanisms that produce free nuclear rotation, similar to the rotation observed in P0 cells.
Nuclear rotation does not occur in spherical par-3 cells at the two-cell stage
It has been proposed that the specialized cortical site drives nuclear rotation in both blastomeres of two-cell par-3 embryos. However, the results above indicate that the cortical site is not required for nuclear rotation in wild-type cells where rotation is under the control of polarity cues. This raises the question of what causes nuclear rotation in par-3 two-cell embryos in the absence of polarity. We have previously shown that nuclear rotation in one-cell par-3 embryos is driven by cell shape (Tsou et al., 2002). To determine whether geometric asymmetry also causes nuclear rotation in par-3 two-cell embryos, we compared centrosome movements in par-3 embryos with normal and altered cell shapes.
To more carefully analyze nuclear rotation in unaltered par-3 embryos, we first traced centrosome movements using live imaging of GFP-tubulin, and RNA interference (RNAi)* to produce the par-3 phenotype in two-cell embryos. As previously reported for par-3(it71) mutants (Cheng et al., 1995), in par-3(RNAi) two-cell embryos, spindles in both cells aligned toward the cell contact region in all embryos (n = 47); this orientation will be referred to as longitudinal (Table I). In 64% of these embryos, longitudinal spindle orientation resulted from an apparently wild-type nuclear rotation where one of the centrosomes moved directly toward the cell contact region (Fig. 3, AE). However, in 36% of the cases (17/47), no nuclear rotation was observed. Rather, the centrosomes were already aligned along the long axis of the embryo as soon as they became visible (Fig. 3, FJ). In such embryos, displacement of the first cleavage spindle resulted in mispositioned centrosomes whose daughters migrated directly onto the longitudinal axis (Fig. 3, GI). Thus, longitudinal spindle orientation in a subset of par-3 embryos is due to abnormal centrosome positioning instead of directed nuclear rotation.
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Nuclear rotation toward ectopic flat sides occurs in par-3 embryos
To further test the hypothesis that cell shape can ectopically orient nuclearcentrosome complexes in the absence of polarity in par-3 cells, additional geometric asymmetry was created by placing embryos on agar pads under coverslips, which results in a flat surface on top of the embryo (Fig. S1). The flatness of the cell contact region was then eliminated by irradiating the adjacent blastomere as described before. In such par-3 embryos, the nuclearcentrosome complex always oriented toward the flat surface/coverslip during prophase (Fig. 3, PS; Table I). Therefore, the spindle set up perpendicular to the flat surface, but as the spindle elongated during anaphase, it then moved within the cytoplasm into a plane parallel to the flat coverslip (Fig. 3, ST). The orientation of the nuclearcentrosome complex toward the flat surface occurred in both par-3 blastomeres tested (AB or P1), and even in those par-3 embryos where the initial centrosome position was longitudinal due to centrosome mispositioning. We conclude that nuclear rotation in two-cell par-3 embryos is entirely driven by the asymmetry of cell shape, which argues against the model that nuclear rotation in par-3 embryos is due to a specific cortical site at the cell contact region or any other intrinsic asymmetry. Further, these results clearly show that cell shape, which does not have any effect on nuclear/spindle positioning in polarized wild-type P1 cells, has a dramatic influence on spindle behaviors in nonpolarized par-3 embryos. This indicates that a PAR polaritydependent mechanism exists in wild-type P1 cells that overrides the effects of geometric asymmetry generated by cell shape, and causes spindles to orient onto the polarized anterior/posterior axis.
PAR-3 is required for wild-type nonpolarized cells to avoid geometry-driven nuclear rotation
As shown above, geometric asymmetry generated by the flat cell contact region or an ectopic flat surface can induce ectopic nuclear rotation in nonpolarized par-3 cells. However, in wild-type AB cells, which are also nonpolarized cells but have PAR-3 present all around the cortex, the flat contact region does not induce ectopic nuclear rotation. These correlations lead to a hypothesis that the uniform distribution of PAR-3 around the cortex is required for wild-type nonpolarized cells to avoid the effects of cell shape, and further, that abnormal PAR-3 localization could inhibit geometry-driven rotation in mutant backgrounds. We have shown that loss of PAR-3 in par-3 mutant embryos causes sensitivity of the nuclear/spindle positioning to the cell shape effect in both one- and two-cell embryos (this paper and Tsou et al., 2002). To further test the hypothesis, we examined the converse situation in which PAR-3 is ectopically expressed. In par-2 mutant embryos, PAR-3 is localized around the entire cortex in the P0 and in both cells of the two-cell embryos (Etemad-Moghadam et al., 1995); our hypothesis predicts that cells in par-2 embryos should be resistant to the effects of geometry. In unaltered par-2(lw32) embryos, nuclear rotation failed half of the time in one-cell embryos (5/11) and failed completely in two-cell par-2 embryos (n = 26; Cheng et al., 1995), consistent with this hypothesis. Further, when the ectopically localized PAR-3 was removed in par-3(it71);par-2(RNAi) embryos, nuclear rotation resumed in both one- and two-cell embryos (n = 11; Cheng et al., 1995). These results suggest that nuclei/spindles in cells with ectopic expression of PAR-3 are resistant to the effects of geometry. To confirm that the nuclear rotation in par-3(it71);par-2(RNAi) embryos is geometry-dependent rotation, we generated spherical cells at the two-cell stage. In such spherical par-3;par-2 cells, nuclear rotation directed toward the cell contact region was never observed (n = 14). The spindles set up transversely in the majority of embryos (10/14). As described earlier for par-3 mutant embryos, in a subset of par-3;par-2 embryos (4/14) spindles formed on the longitudinal axis without nuclear rotation. Finally, in spherical two-cell par-3;par-2 blastomeres with an ectopic flat side created by a coverslip, the nucleus oriented toward the flat side during prophase in all embryos (n = 14) as described earlier for par-3 single mutants. Together, these results support the hypothesis that PAR-3 is required for wild-type AB lineage cells to avoid the negative influence of cell shape that can ectopically orient spindles. Further, these results indicate that ectopic PAR-3 (as seen in par-2 embryos) is inhibiting geometry-driven nuclear rotation rather than the intrinsic mechanism that drives wild-type P1 nuclear rotation. Therefore, we conclude that in contrast to previous interpretations (Cheng et al., 1995), both PAR-2 and PAR-3 are essential for the intrinsically programmed nuclear rotation event that occurs in wild-type polarized P lineage cells. However, neither PAR-3 nor PAR-2 is required for geometry-dependent nuclear rotation, and indeed, PAR-3 somehow inhibits such geometry-dependent rotation.
LET-99 is required for geometry-driven nuclear rotation in par-3 mutant embryos
We showed above that PAR-3 is required for nonpolarized AB cells to be resistant to geometric effects. Based on the behavior of spindles in par-3 mutant embryos, it has been proposed that PAR-3 stabilizes interactions between the astral microtubules and the cell cortex (Etemad-Moghadam et al., 1995; Grill et al., 2001), although the mechanism is unknown. Such a role might be a clue for how PAR-3 functions, directly or indirectly, to resist the effects of geometry in nonpolarized cells as well. To understand more about the novel function of PAR-3 in inhibiting geometry-dependent rotation, we looked for potential candidates that could act downstream of PAR-3. Of the spindle orientation genes described in the literature, only LET-99 has been shown to be localized and/or regulated by PAR-3 (Tsou et al., 2002). LET-99 is uniformly localized around the cortex in par-3 mutants, and the level of cortical LET-99 is higher compared with the levels of cortical LET-99 in wild-type AB cells and par-2 mutant cells (Tsou et al., 2002), where geometry-dependent rotation does not occur. To determine if this ectopic high level of LET-99 is required for par-3 cells to be sensitive to geometric asymmetry, we traced centrosome movements in par-3:let-99 double RNAi embryos. In contrast to the directed rotation toward the cell contact region observed in par-3 embryos, nuclearcentrosome complexes in par-3;let-99 embryos exhibited a random hyperactive rocking motion similar to that described for let-99 embryos (Fig. 4, AF). In some cases, the nuclearcentrosome complex changed 90° in orientation in only 1020 s, and the direction of change appeared completely random (Fig. 4 G). These results indicate that LET-99 is required for par-3 cells to be sensitive to geometric asymmetry, and suggest that having low levels instead of high levels of cortical LET-99 in wild-type AB cells allows them to resist the effects of cell shape.
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Discussion |
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These results challenge the prevailing model for nuclear rotation in C. elegans embryos. The cortical site model proposes that an anterior cortical site captures and shortens microtubules, thus causing nuclear rotation directed toward the anterior cortex of P1 cell (Hyman and White, 1987; Hyman, 1989). This model was first developed based on several lines of evidence. First, during centration and rotation, the nucleus in the P1 cell moves toward the cell contact region, and after rotation, the nucleus becomes closely juxtaposed to the anterior cortex. Second, ablation of the cytoplasm in the area between the centrosomes and the anterior cortex causes rotation to cease; ablation of lateral or lateral posterior areas does not stop rotation. Third, the membrane at the cell contact region invaginates into the P1 cell during rotation, suggesting tension on that part of the anterior cortex. Subsequently, it was found that some components of the dynein associated dynactin complex accumulate transiently in a dot at the anterior cortex (Waddle et al., 1994; Skop and White, 1998), and the model has evolved into one in which the anterior cortical site that captures microtubules is localized at or around the cell division remnant (Waddle et al., 1994; Keating and White, 1998; Skop and White, 1998; Gönczy, 2002). Although dynein and dynactin are required for rotation in the one-cell embryo, it has not been possible to directly test their roles in P1 (Skop and White, 1998; Gönczy et al., 1999). In addition, it has not been shown whether the dot of dynactin or the membrane invagination are a cause or an effect of rotation.
The free rotation that we observed in P1 spherical cells cannot be explained by the presence of a localized cortical site that captures and shortens microtubules because that model predicts the nucleus will move toward the cortical site even in a spherical cell (Fig. 1 A; Hyman, 1989). In addition, we have found that nuclear rotation in par-3 mutant embryos, which lack intrinsic polarity, does not occur when extrinsic cell shape asymmetry is removed. However, nuclear rotation can be induced by introducing ectopic asymmetry via a flat side. Thus, although nuclear rotation in par-3 embryos was previously considered as evidence for ectopic activation of the cortical site in the AB cell, our results clearly indicate that rotation in par-3 embryos is driven by cell shape. Our results also indicate that the membrane invagination associated with the division remnant correlates with a specific cell shape and is not required for centration or nuclear rotation. The observations that the membrane invagination are visible in the irradiated cell in both par-3 and wild-type embryos, and that astral microtubules respond to other cortical cues that position the spindle in the unirradiated spherical cell suggest that UV irradiation has not damaged the cortex. However, whether the cortical site is still present in wild-type spherical P1 cells does not appear relevant. If the cortical site is there, it is not acting as predicted by the model. If the site has been damaged, then the cortical site is not essential because rotation still occurs. Therefore, we conclude that the combination of findings on cell shape effects in both wild-type and par-3 embryos are incompatible with the specialized cortical site model for nuclear rotation.
The occurrence of free rotation in spherical cells suggests that another type of mechanism functions in P1 cells. We propose that nuclear rotation in wild-type one- and two-cell embryos is driven by a programmed polarity-dependent mechanism that causes free rotation, and that the extended movement of the nucleus toward the anterior cortex in P1 cells, as well as the localized invagination of the cortex, are consequences of the physical effects of cell shape. LET-99 is likely to be involved in the polarity-dependent mechanism of rotation because LET-99 is required for nuclear rotation in both P0 and P1, LET-99 is localized in response to PAR-3, and the LET-99 band model predicts free central rotation in spherical cells. This model is consistent with the original laser ablation studies that led to the anterior cortical site model (Hyman, 1989) because the combination of the LET-99 band and cell shape is predicted to result in higher pulling forces directed toward the anterior cortex.
The results of these cell shape studies also significantly change the interpretation of how the PAR proteins control nuclear rotation in wild-type embryos. In contrast to the original model, the findings reported here indicate that PAR-3 and PAR-2 are both required for normal nuclear rotation. Importantly, PAR-3 does not inhibit normal nuclear rotation, but does inhibit geometry-dependent rotation. Our findings also explain previous reports that flattening par-3 embryos resulted in a lower frequency of rotation toward the cell contact region (Cheng et al., 1995). The ectopic flat side of the coverslip would be predicted to compete with the flat cell contact region in directing rotation in such embryos.
A physical model for geometry-dependent nuclear rotation in par-3 embryos
Although the molecular mechanism remains to be determined, it is intriguing that the geometry of two different cell shapes, that of oval cells and cells with one flat/curved-in side, can cause nuclear rotation in par-3 cells. To understand how geometry might work in driving nuclear rotation in cells with no intrinsic polarity, we used several known microtubule properties as assumptions to build a physical model. The first assumption is that microtubules are laterally associated with motors on the cortex, such as dynein, rather than associated end-on. It has been reported that microtubules can interact with the cell cortex either end-on or laterally (Adames and Cooper, 2000). With end-on interactions, microtubule plus ends reach the cortex without bending and are captured by cortical proteins. By depolymerization of microtubules, motile forces can be generated to move objects (Lombillo et al., 1995). The magnitude of forces generated on each microtubule with end-on attachments depends largely on the rate of depolymerization, and will be independent of the angles with which microtubules interact with the cell cortex. Therefore, if the depolymerization rate is uniform around the cortex as predicted for nonpolarized par-3 cells, every microtubule will experience a similar amount of force regardless of the cell shape. With this scenario, geometric asymmetry should have no effect on nuclear rotation and centrosome movements in par-3 embryos. Conversely, if microtubules interact laterally with the cortex, the interaction angle will significantly affect the force vector applied on each microtubule (Fig. 5 A). Therefore, our model is based on lateral attachments. The second assumption is that microtubules tend to bend at small angles due to their stiffness (Gittes et al., 1993).
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Another predicted outcome of the model for cells with one flat side is that the polarized microtubule attachments with dynein will produce an opposite force on the membrane, causing a localized invagination of the cortex at the cell contact region in both par-3 and wild-type P1 cells (Wrinkling effect, Fig. 5 A5), analogous to the wrinkling up of a carpet in a game of tug-of-war. Physically, this wrinkling effect should only occur where the two membranes are not as tightly sealed, such as at the gap present at the cell division remnant (Keating and White, 1998; Skop et al., 2001). Membrane invaginations adjacent to the cell division remnant were previously considered as evidence for the cortical site model in both par-3 and wild-type embryos (Hyman and White, 1987; Hyman, 1989; Waddle et al., 1994; Keating and White, 1998). However, our physical model suggests that invagination could be a consequence rather than a cause of nuclear rotation. The absence of the invaginations in wild-type spherical P1 cells supports this view. Furthermore, the model can also explain why the centrosome is always positioned at the division remnant (the site of the invagination) after nuclear rotation has occurred. In analogy to the tug-of-war game (Fig. 5 A5), if the magnitudes of two dynamically opposing forces are relatively similar to each other, the central position between the two will randomly fluctuate along the surface of the carpet (Fig. 5 A5, top). However, the appearance of a wrinkle on the carpet will dramatically limit the extent of the fluctuation and thus settle and fix the final central position between the two around the wrinkled-up region on the carpet (Fig. 5 A5, middle and bottom). Live imaging of centrosome movements in wild-type embryos has documented such "overshooting" and equilibration to a final position adjacent to the division remnant (Keating and White, 1998). However, in spherical P1 cells, we did not observe rotation oriented toward the division remnant, consistent with the model that the close association of the centrosome in unaltered P1 cells is a consequence of cell shape. The biological meaning for this cell shape-dependent effect of the cell division remnant in fixing the final position of the centrosome is unknown, but our results clearly show that the invagination itself is not required for nuclear rotation and asymmetric spindle elongation in wild-type P1 cells.
PAR-3 regulates spindle orientation in both polarized and nonpolarized cells, potentially through LET-99
In this paper, we have shown that extrinsic geometry can drive ectopic nuclear rotation, which would appear to be problematic for normal development. Polarized cells of the wild-type C. elegans embryo avoid this problem through a PAR polaritydependent mechanism that can override geometric effects and drive wild-type rotation. However, the nonpolarized wild-type AB cell is also resistant to geometric effects and does not show ectopic spindle alignment toward the cell contact region. par-3 mutants are sensitive to geometric affects, indicating that PAR-3 is also required to suppress ectopic geometry-driven rotation in nonpolarized cells. The underlying mechanisms for both may rely on PAR-3's role in LET-99 localization. The localization of LET-99 to a band in the P cells requires PAR-3 and PAR-2, and the absence of the band correlates with failure of nuclear rotation in par-3 and par-2 embryos (Tsou et al., 2002; this paper). In addition, high levels of PAR-3 appear to inhibit localization of LET-99 at the anterior cortex in P cells and at the cortex in AB cells (Tsou et al., 2002). In wild-type AB cells and par-2 mutant two-cell embryos, where PAR-3 is uniformly present and LET-99 is uniformly low, nuclearcentrosome complexes do not orient toward the cell contact region. However, in par-3 embryos where geometry-dependent rotation occurs, LET-99 is present at higher levels at the cortex (Cheng et al., 1995; Tsou et al., 2002; this paper). Furthermore, LET-99 is required in par-3 embryos for the ectopic alignment according to geometry. Although it is possible that loss of LET-99 prevents geometry dependent rotation in par-3;let-99 embryos indirectly, the correlations between PAR-3 levels, LET-99 levels and resistance to geometry are striking. Therefore, we favor the hypothesis that in wild-type embryos, PAR-3 suppresses the effects of cell shape by down-regulating the levels of cortical LET-99 in the AB cell. We propose that it is not only critical that LET-99 be present in a band in cells that undergo nuclear rotation onto the polarized axis, but also for LET-99 levels to be low, but not absent, in other cells to prevent geometry-driven rotation (Fig. 5 B).
In summary, we have found that PAR-3 is essential for the mechanism of nuclear rotation in asymmetrically dividing cells, as well as for avoiding ectopic rotation in nonpolarized cells. The presence of PAR-3 in both types of cells provides a way of regulating the division pattern in multiple cells as a response to embryonic polarity. Such regulation is likely important in other organisms during development, where cells are dividing in multicellular contexts and cell shapes are not spherical. The effects of geometry would need to be modulated in order to produce the normal patterns of division necessary for segregation of cell fate determinants and for morphogenesis.
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Materials and methods |
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RNA interference
Antisense and sense RNAs were transcribed in vitro (MEGAscriptTM; Ambion) from linear DNA templates. Templates were produced by the polymerase chain reaction, using T3 and T7 primers to amplify a partial par-3 cDNA (a gift from K. Kemphues), par-2 cDNA (a gift from L. Boyd; University of Alabama, Huntsville, AL), or a full-length let-99 cDNA. Double-stranded RNAs (1 mg/ml-1; Fire et al., 1998) were injected into adult hermaphrodites, and progeny were analyzed 1624 h later. par-3;par-2 double-mutant embryos were generated by soaking par-3(it71) hermaphrodites in 1.5 mg/ml-1 par-2 dsRNA (Tabara et al., 1998). Wild-type hermaphrodites were simultaneously soaked and examined as a control for effectiveness of par-2(RNAi).
Microscopy and alteration of cell shape
Embryos were mounted unflattened (Rose and Kemphues, 1998a) or flattened with 22 x 22-mm or 22 x 30-mm coverslips on a thin pad of 5% agarose, and examined under differential interference contrast (DIC) or fluorescence optics using time-lapse microscopy. To produce spherical P1 cells in early prophase two-cell embryos, the field diaphragm was closed down to restrict the beam of light to the anterior half of the AB blastomere; the field was then irradiated with UV light (330385 nm) from a 100-W mercury lamp for 37 s. Only those embryos in which P1 was unaffected by irradiation, as judged by normal cell cycle length, were analyzed further. To confirm that mounting embryos on agar pads under coverslips induces an ectopic flat side, a complete Z series (1-µm step) for the top and bottom halves of the same embryo were collected separately on an upright and inverted microscope, respectively (due to the limited working distance of the objective). Images were then manually examined to determine the in-focus area as judged by the appearance of yolk granules. The in-focus information was then used to generate three-dimensional reconstructions with MetaMorph® software (Universal Imaging Corporation).
Online supplemental material
Fig. S1 documents the spherical and flattened shapes of cells. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb200209079/DC1.
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Acknowledgments |
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This work was supported by an American Cancer Society Research Project Grant (00-076-01-DDC) to L. Rose.
Submitted: 16 September 2002
Revised: 28 January 2003
Accepted: 28 January 2003
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References |
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