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Keywords:

  • Drosophila;
  • blastoderm;
  • microfilaments;
  • microtubules

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Dynamic alterations in the spatial organization of cytoskeletal elements constitute a prominent morphological feature of the early, syncytial stages of embryogenesis in Drosophila. Here, we describe and characterize the dynamic behavior of cytoplasmic, subcortical microfilaments, which form a series of nucleus-associated structures, at different phases of the simultaneous nuclear division cycles characteristic of early Drosophila embryos. Remodeling of the cytoplasmic microfilament arrays takes place in parallel to the established cyclic reorganization of cortical microfilament structures. We provide evidence that the cortical and subcortical microfilament populations organize independently of each other, and in response to distinct instructive cues. Specifically, formation of subcortical microfilament structures appears to rely on, and spatially mirror, the organization of polarized microtubule arrays, while cortical microfilament restructuring constitutes a centrosome-dependent process. Genetic analysis identifies a requirement for SCAR, a key mediator of Arp2/3-based microfilament dynamics, in organization of subcortical microfilament structures. Developmental Dynamics 236:662–670, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Dynamic spatial organization of the microfilament-based cytoskeleton, composed primarily of bundled filament configurations and inter-twined filamentous networks, presents a key mechanism through which cells control their shape and locomotion, and regulate cortical and cytoplasmic events. Cell-surface–associated protein complexes, signaling elements, and cross-linkers are recognized as the major contributors to the establishment, maintenance, and dynamic behavior of microfilament arrays (see Revenu et al.,2004, for a recent review). Demonstrations of a direct influence of cytoskeletal elements on microfilament organization are, however, relatively scarce. In this context, we report here on a novel aspect of cytoplasmic microfilament dynamics in early Drosophila embryos, for which microtubules appear to play a key instructive role.

The early stages of embryogenesis in the fruit fly, Drosophila melanogaster, provide an attractive setting for assessing various aspects of cytoskeletal organization, in the context of a developing organism (Foe et al.,1993; Schejter and Wieschaus,1993; Sullivan and Theurkauf,1995). Early Drosophila development is characterized by 13 rapid and synchronous rounds of nuclear division, that proceed without intervening cytokinesis (Zalokar and Erk,1976; Foe and Alberts,1983). These divisions initially occur in the interior of the embryo, while the final four cycles—termed the “syncytial blastoderm” stage—take place after migration of the nuclei to the cortex, where they arrange in a subcortical monolayer. Major roles for the microtubule and microfilament-based cytoskeletons have been characterized, both in overseeing the stepwise, concerted motion of several hundred migrating nuclei (Baker et al.,1993; von Dassow and Schubiger,1994), and subsequently, in organization of the subcortical nuclear array. A key aspect of the latter entails cyclic restructuring of cortical actin-based structures (Karr and Alberts,1986; Foe et al.,2000), which, in the absence of membrane partitions, act to maintain a uniform distribution of nuclei within the shared cytoplasm.

We re-examined cytoskeletal organization in the syncytial blastoderm embryo, with the primary purpose of assessing the relative spatial distributions of microtubules and microfilaments. Our analysis distinguishes between the well-described cortical structures and arrays of subcortical cytoplasmic microfilaments, which appeared to associate with spindle and astral microtubules. Genetic analysis and drug-induced alterations suggest that the cortical and cytoplasmic F-actin populations behave independently of each other. Significantly, dynamic restructuring of the subcortical microfilament arrays in syncytial Drosophila embryos—but not of cortical microfilament structures—appears to provide a novel instance in which the microtubule-based cytoskeleton dictates the spatial organization of associated microfilaments.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Dynamic Organization of Cortical and Cytoplasmic Microfilaments in the Drosophila Syncytial Embryo

We used rhodamine–phalloidin staining to reveal the spatial distribution of microfilaments in Drosophila syncytial blastoderm stage embryos (Fig. 1). The dynamic, nuclear cycle-dependent restructuring of cortical microfilaments during this developmental stage is well described (Warn et al.,1984; Karr and Alberts,1986; Foe et al.,2000) and includes formation of actin “cap” structures that overlie interphase nuclei, and transient, furrow-like invaginations that separate dividing nuclei at mitosis (Fig. 1A,B). Dynamic transitions between the cortical cap and furrow configurations are associated with all four syncytial cortical divisions preceding blastoderm cellularization. The corresponding organization of cytoplasmic microfilaments was visualized by optical sections through the subcortical region, at a depth of 5–10 μm from the surface (Fig. 1C–E). As reported in studies of preblastoderm embryos (Karr and Alberts,1986; von Dassow and Schubiger,1994), cytoplasmic microfilaments are arranged in a network of thin fibers, interspersed with sparse actin aggregates (Fig. 1C). The spatial distribution of the fibrous network is not uniform, however, with elevated concentrations of fibers in the vicinity of the nuclei. The nucleus/spindle-associated microfilament network intensifies and persists throughout the syncytial blastoderm divisions (Fig. 1D,E), although details during later division cycles become difficult to discern, due to the “crowded” distribution of syncytial nuclei.

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Figure 1. Cortical and cytoplasmic distribution of microfilaments in the Drosophila syncytial blastoderm embryo. In this and subsequent figures, microfilaments are visualized by rhodamine–phalloidin staining and microtubules with anti-tubulin antibodies. A,B: Cortical microfilament structures in cycle 11 embryos. Individual F-actin caps (A) overlie nuclei at interphase, while microfilaments line the honeycomb pattern of transient cortical furrows during mitosis (B). C–E: Cytoplasmic microfilament organization at the subcortical level in syncytial blastoderm embryos. Subcortical microfilaments are found to organize into distinct perinuclear concentrations, once nuclei (n) penetrate the cortical region at cycle 10 (C). The cytoplasmic F-actin distribution at this stage consists of thin fibers and dispersed aggregates. D,D″: Simultaneous visualization of microtubules (D and green in D″) and microfilaments (D′ and red in D″) during anaphase of a cycle 10 embryo, shows that practically all phalloidin-stained material is either concentrated around the dividing nuclei or present in dispersed aggregates. E,E″: A cycle 11 embryo at metaphase displays a similar distribution of cytoskeletal elements. Scale bars = 10 μm.

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Subcortical Microfilament Structures Are Associated With Astral Microtubules

A detailed comparative analysis during division cycle 10, the first cortical division cycle, revealed a close correspondence between the spatial distribution of subcortical microfilaments and the nuclear-cycle organization of microtubules (Fig. 2). Microfilaments are closely associated with the interphase nuclei during cycle 10, surrounding their entire circumference, and extending both toward and away from the plasma membrane (Fig. 2A,A′). Centrosome migration to the opposite sides of the nucleus and formation of an elongated spindle at prometaphase were accompanied by a corresponding elongation of the microfilament array (Fig. 2B,B′,B″). Remarkably, distinct, aster-like F-actin accumulations were observed in close correspondence to both spindle poles. Both the spindle region and aster-like arrays of microfilaments became more pronounced with progression to metaphase and early anaphase (Fig. 2C,C′). This enhancement of spindle-level microfilaments is accompanied by appearance of a strong accumulation of F-actin at the center of the aster-like microfilament array. Double-staining experiments demonstrated colocalization of this focal point with the centrosomal antigen CP190 (Fig. 2D,D′,D″), suggesting a close spatial relationship between the centrosome and the focus of the microfilament aster.

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Figure 2. Spatial correspondence of microtubules (MT) and subcortical microfilaments (MF) during division cycle 10. A,A′: Microfilament networks (arrows) associate with and surround cycle 10 interphase nuclei. B,B′: As nuclei enter mitosis, distinct microfilament accumulations are observed at the spindle poles (arrows). B″: Colored merged image of B and B′, with microtubules in green and microfilaments in red, underscores spindle-pole location of microfilament clusters. C,C′: The spindle-enveloping and aster-like microfilament distributions are accentuated at metaphase of cycle 10. As previously described (Karr and Alberts,1986), cortical microfilaments form flat caps (c) above the spindles rather than furrows, during mitosis in cycle 10 embryos. D,D′: Double staining of a dividing cycle 10 embryo for the centrosomal antigen CP190 (D) and F-actin (D′). D″: The merged image, with the CP190 staining colored green and the microfilaments colored red, demonstrates that the spindle pole is positioned at the core of the microfilament aster-like structure. E,E′: The microfilament aster structure becomes diffuse during anaphase of cycle 10. F,F′,F″: At the end of mitosis, microfilaments are no longer associated with the spindle midbody remnants (mb), and once again surround the cycle 11 interphase daughter nuclei. Scale bar = 10 μm.

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Progress through the final phases of division cycle 10 is associated with further re-distribution of the microfilaments within the subcortical array. The focal accumulation at the center of the aster-like microfilament array becomes diffuse by late anaphase (Fig. 2E,E′), whereas the spindle-enveloping microfilaments disassemble at telophase (not shown). In contrast, the aster-like microfilament arrangements increase in size, eventually surrounding the daughter nuclei of the cycle 10 division (Fig. 2F,F′,F″). As in interphase of the previous cycle (Fig. 2A,A′), these microfilament arrays are in close spatial correspondence to the interphase microtubule asters, leaving almost free the region occupied by the spindle mid-body remnants.

Further demonstration of the close association between microfilaments and astral microtubules was obtained from study of the cycle 10 division in embryos derived from centrosomin (cnn) mutant females. The cnn locus encodes a core centrosomal protein required for the structural integrity of Drosophila microtubule organizing centers (Heuer et al.,1995; Vaizel-Ohayon and Schejter,1999; Megraw et al.,2001). Early embryos lacking cnn function possess only rudimentary centrosomes and display a variety of defects in organization of spindle microtubules (Megraw et al.,1999; Vaizel-Ohayon and Schejter,1999). In keeping with previous observations, our current analysis confirmed the variable nature of the cnn early embryonic mutant phenotype, identifying both embryos with biastral spindles (Fig. 3A,B) and embryos with barrel-shaped anastral spindles (Fig. 3C). The polarized microtubule arrays found at the biastral spindle poles in cnn mutant embryos display a variety of abnormalities, including different degrees of spindle-pole detachment and unevenness of spindle-pole size. Shifting of microtubule asters away from the spindle regions resulted in a closely matching displacement of the aster-like microfilament arrays (Fig. 3A,A′). Furthermore, the robustness of these arrays mirrored the size variations of the spindle-pole arrays of microtubules (Fig. 3B,B′). Finally, in cnn embryos displaying barrel-shaped anastral spindles, large holes formed in the subcortical microfilament layer corresponding to the spindle regions, but aster-like arrangements were never observed (Fig. 3C,C′). These observations provide a compelling demonstration of the spatial correspondence between astral arrays of microtubules and remodeling of subcortical microfilaments.

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Figure 3. The position and size of subcortical microfilament structures mirror those of astral microtubules in cnn mutant embryos. Panels show the spatial pattern of microtubules (MT, left column) and microfilaments (MF, center column) during mitosis of cycle 10 (all cnn embryos shown in this study were derived from cnnHK21/Df(2R)8-104 females (Vaizel-Ohayon and Schejter,1999)). In the merged images (right column), microtubules are colored green and microfilaments are in red. A,A″: Microfilament aster-like structures remain in close correspondence to shifted or detached microtubule asters (arrows). B,B″: The size of the microfilament aster-like structures appears to match the variably sized microtubule asters. C,C″: Distinct subcortical microfilament concentrations are missing from the vicinity of anastral spindles in cnn embryos. Scale bar = 10 μm.

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Subcortical Microfilament Organization Relies on Polarized Microtubule Arrays, While Cortical Microfilament Dynamics Depend on Intact Centrosomes

cnn mutant embryos display abnormalities in both centrosome structure and in the emanating astral microtubule arrays, precluding a clear determination of the malfunction responsible for the defects in subcortical microfilament organization. To address this issue, we sought to engineer circumstances where centrosome and astral microtubule functions are uncoupled and can be assessed independently. We first analyzed the effects of colchicine on microfilament organization. Application of this inhibitor results in depolymerization of all detectable microtubules in the syncytial embryo but does not affect localization of “core” centrosomal proteins such as gamma-tubulin, CP190, and Centrosomin (Raff et al.,1993; Oegema et al.,1995; Vaizel-Ohayon and Schejter,1999), thereby allowing us to follow microfilament dynamics in a microtubule-free environment, in which centrosomes remain intact.

Treatment of wild-type embryos with colchicine at cycle 9, just before the completion of nuclear migration, strongly affected the organization of subcortical microfilaments shortly thereafter, as nuclei penetrated the cortical region (Fig. 4A). While a general subcortical F-actin network persists, it completely lacks the centrosome and nucleus-associated structural features characteristic of this stage (Fig. 4A,A′). F-actin aggregates are greatly reduced in number as well. Repeating this experiment on syncytial stage, cycle 11 embryos, in which nuclei were already positioned at the cortex, allowed us to simultaneously assess and compare the effects of microtubule depolymerization on both the subcortical and cortical microfilament arrays. As reported elsewhere (Stevenson et al.,2001), colchicine-treated syncytial embryos arrest during mitosis. Cortical actin in these embryos reorganized properly after microtubule disruption, as demonstrated by formation of mitotic furrows between adjacent arrested nuclei (Fig. 4B; compare with Fig. 1B). Furrows appeared cytologically normal and largely intact close to the embryo surface (Fig. 4B), although they did not extend uniformly between the nuclei and reached a variable depth (Fig. 4C). In marked contrast, the subcortical network of microfilaments loses all aspects of organization after microtubule depolymerization (Fig. 4C), displaying a dispersed spatial distribution. Significantly, both the aster-like arrays of microfilaments that surround the centrosomes and the actin network that envelopes the nuclear regions during the syncytial blastoderm division cycles (Figs. 2, 3) are absent. These observations suggest a primary influence for microtubules on organization of subcortical microfilaments, but only a minor role in controlling cortical microfilament dynamics.

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Figure 4. Cortical and subcortical microfilament organization are influenced by distinct instructive cues. A–C: Microfilament organization in colchicine-treated embryos. A: Centrosomes (visualized with anti-CP190) mark the position of spindle-poles, in a cycle 10 embryo treated with colchicine during the final phase of nuclear migration. A′: Subcortical microfilaments in this embryo fail to organize and display a homogeneous distribution (compare with Fig. 2D,D″). B,C: A cycle 11 embryo treated for 10 minutes with colchicine before fixation and staining. B: An optical section just below the cortex demonstrates the proper restructuring of cortical microfilaments (CMF) into mitotic furrows, in the absence of microtubules (see also Stevenson et al.,2001). C: A deeper optical section reveals discontinuities in the mitotic furrow network. This level corresponds in depth to the position of the nuclei and to the subcortical microfilament network (SCMF), which occupies the cytoplasm between the furrows, and now appears dispersed and devoid of characteristic structural features. D,E: Cortical microfilament organization in spg mutant embryos. Phalloidin staining of embryo progeny of spg242/Df(3R)3450 mutant females demonstrates lack of organization of cortical microfilaments throughout syncytial stage nuclear divisions. Embryos at interphase of cycle 12 (D) and mitosis of cycle 11 (E) are shown. F,F′″: Subcortical microfilament organization in spg mutant embryos. F: Anti-tubulin staining reveals the position and orientation of nuclei undergoing mitosis in a cycle 11 spg242/Df(3R)3450 mutant embryo. F′″: Cortical microfilaments above the nuclei (F′) fail to organize into coherent furrow structures, similar to panel 4E. The subcortical network, however, appears unaffected by the mutation, displaying structural features similar to those present in wild-type embryos (arrows and merged image, F′″). G,G″: Microfilament organization near polar bodies. G: An astral arrangement of microtubules (arrow) is associated with polar bodies in the cortical region of an early Drosophila embryo. G′: An aster-like configuration of microfilaments (arrow) is positioned on the internal aspect of the microtubule array. Scale bars = 10 μm.

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Further support for the notion that the cortical and subcortical microfilament networks respond to distinct instructive cues, comes from analysis of microfilament organization in sponge (spg) mutant embryos. The primary phenotypic feature of syncytial embryos derived from homozygous spg mothers is a disruption of the spatial organization of cortical actin (Postner et al.,1992). In keeping with previous studies, we observed that both cortical actin caps and mitotic furrows fail to form in syncytial blastoderm stage spg embryos. Instead, cortical microfilaments retain the generally uniform, unstructured appearance of preblastoderm stages, with the exception of a reduced distribution directly above the dividing nuclei (Fig. 4D,E,F,F′). In contrast to the dramatic effect on the structuring of cortical actin, all of the hallmarks of subcortical microfilament reorganization are observed in spg embryos, including formation of a microfilament shell surrounding the spindle region, and spreading out of aster-like microfilament arrays, in close spatial correspondence to the microtubule asters (Fig. 4F,F′,F″,F′′′).

To investigate whether intact microtubule organizing centers are essential for spatial modeling of the subcortical actin network, we examined the distribution of microfilaments near polar body nuclei. This distinct group of three nuclei, which originate from the haploid complements “left over” at completion of meiosis of the oocyte nucleus, persist in metaphase-like configurations until nuclear division cycle 10–11 near the embryo surface (Foe et al.,1993). The chromosomes of polar body nuclei are arranged in a “starburst” configuration, within a large astral array of microtubules organized in the absence of a true centrosome (Foe et al.,2000; M.G.R and G.C., unpublished observations). Double labeling for microfilaments and tubulin revealed an aster-like array of microfilaments in close correspondence to the polar body microtubules (Fig. 4G,G′, G″; see also Foe et al.,2000). These observations suggest that ordered microtubule arrays are capable of organizing subcortical actin in the early embryo, even in the absence of centrosomes.

Organization of the Subcortical Microfilament Network Requires SCAR

Modeling of the subcortical microfilament network by nuclei and their associated microtubule arrays may operate by means of reorganization of existing microfilaments or, alternatively, through de novo actin filament polymerization. An initial indication that the latter mechanism is relevant comes from analysis of microfilament dynamics of SCAR mutant embryos. The SCAR locus encodes the single Drosophila homolog of Scar/WAVE proteins, and serves as the primary mediator of the microfilament nucleating Arp2/3 complex in Drosophila (Zallen et al.,2002). While both Arp2/3 subunits and SCAR are required during oogenesis (Hudson and Cooley,2002; Zallen et al.,2002), germline clones homozygous for the weak SCARk13811 allele produce fertilizable eggs, allowing for assessment of SCAR function during embryogenesis. Indeed, a partial requirement for SCAR and Arp2/3 during formation of cortical actin structures has been described, in that interphase caps fail to expand and reorganize into furrows during mitosis (Stevenson et al.,2002; Zallen et al.,2002). We now report a dramatic effect of mutations in SCAR on the organization of subcortical microfilaments (Fig. 5A,A′,A″). While the amount of subcortical actin aggregates appears unaffected in SCARk13811 mutant embryos, the actin network surrounding the nuclear regions is strongly reduced, and aster-like microfilament concentrations are rare. Given the hypomorphic nature of this allele, we expect that further reduction in SCAR function would fully abolish both the spindle-associated and astral microfilament distributions. These observations contrast with the generally normal distribution of cytoplasmic microfilaments in syncytial embryos, in which function of diaphanous (dia), encoding a second major modulator of microfilament dynamics (Afshar et al.,2000; Grosshans et al.,2005), has been impaired (Fig. 5B,B′,B″). We, therefore, envisage a key role for polymerization of new actin filaments by means of the Arp2/3 complex, specifically mediated by SCAR, in cytoplasmic microfilament dynamics of syncytial stage Drosophila embryos.

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Figure 5. A,A″: Subcortical microfilaments fail to organize in SCAR mutant embryos. Anti-tubulin (A) and phalloidin (A′) staining of a SCARk13811 germline clone-derived embryo identify the position and orientation of dividing nuclei during mitosis of cycle 11 and reveal the absence of associated subcortical microfilament structures. Cortical microfilaments in such embryos display a cap-like configuration during mitosis (Zallen et al.,2002). B,B″: Microfilaments properly associate (arrows in B′) with the spindle poles of a similarly stained dia5 germline clone-derived embryo.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The present study reveals a novel aspect of the dynamic behavior exhibited by the microfilament-based cytoskeleton in syncytial blastoderm stage Drosophila embryos. In addition to the well-described cyclic restructuring of cortical microfilaments characteristic of this early phase of embryogenesis, we have documented nuclear-cycle–dependent organization of cytoplasmic microfilaments, at the subcortical level occupied by a layer of synchronously dividing nuclei. Remarkably, subcortical microfilaments adopt a series of spatial configurations that mimic the structural alterations exhibited by nucleus-associated microtubules. Thus, a microfilament array surrounds interphase nuclei and matches the radiating spatial distribution of microtubules within the interphase aster. As microtubules reorganize to form a mitotic spindle, subcortical microfilaments are found to envelope the spindle and to concentrate in a pair of aster-like structures, centered on the spindle poles. Our analysis suggests several principles that guide these dynamic rearrangements:

First, our observations are consistent with an instructive role for microtubules in directing cytoplasmic microfilament dynamics. Abnormal or even complete loss of subcortical microfilament organization is a common consequence of both epigenetic and genetic perturbations of the microtubule-based cytoskeleton. A prominent example is the close correspondence in both size and position that exists between the variable spindle-pole microtubule asters found in cnn mutant embryos, and the aster-like microfilament arrays associated with them. Moreover, microfilament aster-like arrays fail to form altogether next to anastral spindles in cnn embryos, or after depolymerization of microtubules by treatment with colchicine. We have further observed that astral microfilament arrays form next to the meiotic remnants known as “polar bodies,” a chromatin-containing structure embedded within a microtubule aster, which lacks a centrosomal core (see also Foe et al.,2000). Thus, polarized microtubule arrays appear not only necessary for restructuring of subcortical microfilaments, but sufficient to act in this capacity, even in the absence of a conventional microtubule organizing center.

Second, dynamic rearrangements of syncytial blastoderm cortical and subcortical microfilament arrays occur simultaneously, but in response to distinct instructive cues. While subcortical microfilament dynamics appear to be driven, as discussed above, by polarized microtubule arrays, a variety of studies have demonstrated that cortical microfilament organization requires centrosomes (Raff and Glover,1989; Yasuda et al.,1991; Vaizel-Ohayon and Schejter,1999). Centrosomes perform this function even in the absence of associated microtubules (Stevenson et al.,2001; and this study). The failure of centrosomes to promote cytoplasmic microfilament organization in such a microtubule-free environment, coupled with the formation of aster-like microfilament structures near polar bodies, suggest that centrosome influence is restricted to organization of cortical microfilaments. Additional evidence for distinct mechanisms underlying organization of the cortical and subcortical microfilament arrays comes from our analysis of spg mutant embryos, in which cortical microfilament structures fail to form, but subcortical microfilaments display all aspects of the restructuring characteristic of syncytial blastoderm stages in wild-type Drosophila embryos.

Third, demonstration of a direct role for SCAR—and thus presumably, for the functionally associated Arp2/3 complex—strongly suggests that de novo actin filament formation, using microtubule arrays as polymerization guides, plays a key role in establishment of the cytoplasmic, nucleus-associated microfilament arrays.

The close, instructive association described here between microtubule and microfilament structures should be considered in the context of established interactions between these major cytoskeletal systems. The significance of such interactions to fundamental cellular processes such as locomotion, trafficking, and morphogenesis is widely recognized (Goode et al.,2000; Rodriguez et al.,2003; Etienne-Manneville,2004). The primary underlying mechanisms appear to rely on elements such as signal transduction factors and motor proteins, which function as indirect mediators between independently constructed microtubule and microfilament arrays. A particularly well-studied process in this context is the manner by which the microtubule-based mitotic spindle dictates the cell-membrane site at which a contractile acto-myosin ring will form and execute the final phase of cytokinesis (Glotzer,2005), although even in this instance, the underlying mechanism is yet to be fully elucidated (Burgess and Chang,2005). In addition to such indirect interactions, persuasive evidence demonstrates that microtubules and microfilaments are capable of directly influencing each other's distribution, thereby affecting the spatial organization of the cognate filamentous system (Waterman-Storer et al.,2000; Schaefer et al.,2002). The results of the present study contribute to this emerging field by revealing an in vivo setting, where the capacity of microtubules to serve as a structural template for remodeling the microfilament-based cytoskeleton has been used.

The functional significance of microtubule-dependent cytoplasmic microfilament structures in Drosophila syncytial embryos is currently an open question. The genetic approaches available in the Drosophila system hold the promise that specific disruption of subcortical microfilament structures may be achievable. Indeed, our observation that subcortical microfilament organization is strongly disrupted in SCAR mutant embryos provides an initial inroad along this route. Studies of nucleus-associated microfilament arrays in other systems have identified two major potential roles. The first is that microfilaments can contribute to the structural integrity of nuclei, as suggested for the intranuclear actin mesh present in Xenopus oocytes (Bohnsack et al.,2006). Alternatively, as suggested in previous studies (Silverman-Gavrila and Forer,2000; Sampson and Pickett-Heaps,2001) and supported in compelling fashion recently (Lenart et al.,2005), nucleus-associated microfilaments can act to ensure the fidelity of spindle dynamics and chromosome distribution during and after mitosis. After preliminary examination, which failed to detect abnormalities in the gross structure of nuclei during the syncytial blastoderm divisions in SCAR embryos, we favor the latter as a more likely role for the nucleus-associated microfilament structures described above. Further, detailed analysis of mitosis in SCAR mutant embryos should prove informative in assessing the veracity of this and other ideas, for the functional requirements that necessitate the unique spatial organization of cytoplasmic microfilaments in early Drosophila embryos.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Drosophila Genetics

Oregon-R flies were used as the strain for producing wild-type embryos (n = 83). cnn (n = 77, 23 with biastral spindles, 44 lacking asters) and spg (n = 61) mutant embryos were derived from females hemizygous for strong alleles of these maternal-effect mutations (cnnHK21/Df(2R)8-104 [Vaizel-Ohayon and Schejter,1999] and spg242/Df(3R)3450 [Postner et al.,1992], respectively). To obtain embryos deficient in SCAR (n = 55) or dia (n = 42) activity, germline clones were produced in SCARk13811, FRT40A/CyO and dia,5 FRT40A/CyO heterozygous females, respectively, as described (Afshar et al.,2000; Zallen et al.,2002; Grosshans et al.,2005).

Embryo Preparation and Visualization by Immunofluorescence and Confocal Microscopy

Embryos (0- to 2-hr-old; at 24°C) were dechorionated in a 50% bleach solution for 2–3 min, rinsed in distilled water, dried on filter paper, and transferred to one of two fixation solutions. To simultaneously visualize microfilaments and centrosomes, embryos were fixed by rapid shaking for 30 min on the interphase of a 1 ml:4 ml mixture of 4% paraformaldehyde (methanol-free, Pella Scientific) and heptane. Embryos destined for simultaneous visualization of microtubules and microfilaments were similarly fixed, except that the paraformaldehyde portion of the fixative was replaced by 4% formaldehyde. Fixed embryos were transferred to a small drop of phosphate buffered saline (PBS) on double-sided tape, and the vitelline envelope was removed manually with thin needles. Devitellinized embryos were then transferred to a 1% Triton X-100 in PBS solution for 10 min, rinsed for 10 min in PBS, and incubated for 30 min in PBS+0.1% bovine serum albumin (BSA; PBS-BSA). Embryos were then incubated with the primary antibody (rabbit polyclonal anti-CP190 serum Rb188 [Whitfield et al.,1988] diluted 1:400 in PBS-BSA, or mouse monoclonal anti–β-tubulin [Boehringer Mannheim, UK] diluted 1:200), for 1 hr at room temperature. After washing in PBS-BSA, the embryos were incubated for 30 min with the appropriate fluorescein-coupled (goat) secondary antibody (Cappel, West Chester, PA), diluted 1:600 in PBS-BSA. Microfilaments were visualized in both cases with 10 units/ml rhodamine–phalloidin (Molecular Probes), which was added either to the primary antibody (centrosome) or secondary antibody (microtubule) incubations. Stained samples were rinsed for 20 min in PBS and mounted in small drops of 90% glycerol containing 2.5% n-propyl-gallate.

Digital optical sections of whole-mount embryos were produced using a Leica TCS 4D laser scanning confocal microscope equipped with an Argon–Krypton mixed gas laser and coupled to a Leica DMRBE microscope equipped with a 63× PL Apo 1.4 objective (Leica Lasertechnik, Heidelberg). For double stainings, the images of the two fluorochrome distributions were recorded separately by averaging 8–16 scans of a single optical section to improve the signal/noise ratio, keeping laser emission low to attenuate photobleaching. Images collected at several focal planes were superimposed, merged into a single file, and imported into Adobe Photoshop to adjust size and contrast.

Drug Treatment

For the microtubule depolymerization experiments, 2- to 3-hr-old embryos (n = 69) were dechorionated in a 50% bleach solution, washed in distilled water, dried on filter paper, and permeabilized with heptane as described (Limbourg and Zalokar,1973). The eggs were then quickly dried, covered with tissue paper, and incubated with 5 mM colchicine (Sigma) in D20 medium (Echalier and Ohanessian,1970) for 10 or 20 min at 24°C. The tissue paper prevents the embryos from floating on the surface of the solution and ensures that the entire embryo is in contact with the drug solution. Identical incubations in D20 medium without colchicine were performed and monitored, to ensure that the described phenotypes were indeed a result of treatment with the inhibitor.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Haya Avital from the WIS Graphics Department with help in image processing and figure composition. G.C. was funded by grants from PRIN and PAR (University of Siena), and E.D.S. was funded by the Israel Science Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES