BIOLOGICAL OVERVIEW

Lava lamp (Lva) is nostalgically named for the apical/basal movements observed in the Golgi bodies of Lva mutants during the process of cellularization, reminiscent of the motion of droplets in a lava lamp (Sisson, 2000). Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, a biochemical screen was conducted for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. A novel MMAP, Lava Lamp is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Functional analysis shows that cellularization is dramatically inhibited upon injecting embryos with anti-Lva antibodies (IgG and Fab). In addition, brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin (see alpha Spectrin) and CLIP190. It is suggested that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. These results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion (Sisson, 2000).

Drosophila cellularization is a dramatic variation of animal cell cytokinesis. It transforms a one-cell syncytium into a multicellular embryo by simultaneously encapsulating roughly 5,000 cortical nuclei with one continuous plasma membrane furrow. The furrow invaginates between adjacent nuclei perpendicular to the cell surface, and then widens along its base to seal each nucleus off from the inner cytoplasm. Although the honeycomb-patterned cellularization furrow appears distinct from the ring-like cytokinesis furrow, the mechanisms underlying their formation are strikingly similar. The progression of each furrow depends on an actomyosin-based contractile apparatus associated with the leading edge of each furrow. In addition to microfilaments (F-actin) and myosin II, each contractile apparatus also consists of the putative stabilizing elements, Anillin, Septins (see Peanut), and formin homology proteins. Furrow progression in each case also appears to rely on the export of intracellular membrane to the cell surface (Sisson, 2000 and references therein).

While significant progress has been made toward understanding the assembly and function of the contractile apparatus, much less is known about the mechanism of membrane export. In Xenopus, it is well established that membrane is delivered during anaphase to the furrowing portion of the plasma membrane, but the exact target along the furrow is uncertain. At least some of the membrane is Golgi derived; however, other sources of membrane have also been implicated. Regardless of the intracellular membrane source, microtubules (MTs) appear to be required for its delivery. In the case of cellularization, neither the membrane source nor the modes of membrane delivery are known. However, the need for membrane export during cellularization is underscored by an increase in cell surface area, and the requirement for Syntaxin 1 and Dynamin, proteins implicated in membrane transport (Sisson, 2000 and references therein).

During both cellularization and cytokinesis, the coordination of the F-actin and MT cytoskeletal systems contributes to contraction and/or membrane export. Although the precise mechanism of this dependency relationship is unknown, MTs may act as tracks for recruiting factors required for the establishment and/or maintenance of the contractile ring, or provide structural stability to the contractile ring late in cytokinesis. During cellularization, long MTs grow out from apically positioned centrosomes down over the surface of nuclei and project their plus ends into the embryos interior, forming 'inverted baskets' around each nucleus. Throughout most of cellularization, the contractile apparatus appears to move down along these MTs as it leads the furrow inward at its tip. When embryos are treated with drugs that destabilize either F-actin or MTs during cellularization, furrow progression is severely disrupted. The extent to which these treatments disrupt contraction versus membrane export is unknown (Sisson, 2000 and references therein).

An effort was undertaken to identify additional proteins involved in cellularization in order to better understand the mechanisms underlying cellularization and cytokinesis. Because the F-actin and MT cytoskeletal systems appear to intimately associate during cellularization, binding studies were initiated to identify microfilament/microtubule-associated proteins (MMAPs) from Drosophila embryo extracts. This approach combines biochemical screens for F-actin- and MT-binding proteins in Drosophila. Twenty-one MMAPs were isolated in this study. Of these, 17 were identified and include seven proteins previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Dynamin A and B, Peanut, Septin 1, and Septin 2. Also isolated was CLIP190, a known partner of Myosin heavy chain at 95F (Jaguar). CLIP190 is the Drosophila homolog of human CLIP170, a protein implicated in linking vesicles and organelles to MT plus ends. In addition, a novel protein, Lava Lamp (Lva), was identified. Functional analysis of Lva indicates that it too is required for cellularization. Lva is Golgi associated and may function as part of a Spectrin-based scaffold. The results suggest that MT-dependent, Golgi-derived membrane vesicle export is a key mechanism of Drosophila cellularization (Sisson, 2000).

Golgi bodies are found positioned at the tips of cellularization furrows in fixed embryos suggesting that some Golgi might specifically associate with the furrow. To assess this possibility, Golgi was observed in live cellularizing blastoderms using scanning confocal movies. Rabbit anti-Lva antibodies were diluted, indirectly labeled with anti-rabbit-Cy5 fluorescent antibodies, and injected into embryos at the start of cellularization. When the Cy5-tagged anti-Lva antibody is injected, Cy5 fluorescence quickly assumes a punctate distribution at the basal ends of nuclei. Neither diluted antibody inhibits furrow progression. After the fixation of embryos injected with anti-Lva/Cy5 antibodies, the Cy5-marked puncta were found to perfectly colocalize with Golgi p120 by immunofluorescence, indicating that the Cy5-marked puncta are indeed Golgi bodies. Although apically positioned Golgi bodies are not readily visible in sagittal views, grazing optical sections clearly show Cy5-marked Golgi bodies position apically of nuclei. The large amount of background Cy5 fluorescence appears to obscure the apical Golgi bodies when viewed sagittally (Sisson, 2000).

The Cy5-marked Golgi undergo dramatic movements and associate with the furrow front during cellularization. Cellularization begins during the 14 syncytial nuclear cycle. Initially, the furrow progresses slowly ('slow phase'), and then, ~40 min into nuclear cycle 14, the rate suddenly increases until the end of cellularization ('fast phase'). During the first 5-15 min of cycle 14, the Cy5-marked Golgi move in a saltatory manner at the base of nuclei. At ~20 min, individual Golgi bodies move apically and become closely associated with the furrow front, accumulating there over the next 10-20 min of the slow phase. Throughout the first 20-25 min of the fast phase, the Cy5-marked Golgi bodies remain intimately associated with the advancing furrow front. When the furrow front has progressed 5-8 µm beyond the basal ends of the nuclei, Golgi reverse their direction and move rapidly toward the cell surface again, increasing the total amount of Golgi within the apical cytoplasm. These apical/basal movements of the fluorescently marked Golgi bodies are identical to the movements of comparably sized organelles observed in living embryos by Nomarski DIC optics, and resemble the motion of droplets in a lava lamp (Sisson, 2000).

The trajectory of the Golgi movements suggested it might be MT based. To test this possibility, Golgi bodies were fluorescently marked, and then after MT inverted-baskets formed, embryos were reinjected with 25 mM colchicine either 15 or 25 min into cycle 14 to depolymerize MTs. When colchicine is injected at 15 min, Golgi fails to move apically, instead accumulating along the basal ends of nuclei, and furrow progression was severely disrupted. In embryos injected with colchicine at 25 min, Golgi bodies move apically during the slow phase, but fail to do so during the fast phase, and furrow progression is only mildly effected. Although furrow progression is severely inhibited upon injecting the F-actin destabilizing drug cytochalasin D early in cycle 14, Golgi bodies still move apically during the slow phase, as they do during the fast phase upon later injections that minimally affect furrow progression. The organelles seen by Nomarski DIC optics display the identical pattern of MT-dependent, F-actin-independent movements in live cellularizing blastoderms. It is believed these particles are likely to be Golgi bodies (Sisson, 2000).

Because no mutations are known to exist in the lva gene, a concentrated preparation (3.8 mg/ml) of the anti-Lva antibody was injected into embryos at the start of cycle 14 to block Lva function, and the effects on Golgi bodies and furrow progression were observed. To visualize the furrow front in these experiments, embryos bearing a spaghetti squash (sqh)-green fluorescent protein (gfp) transgene were observed. sqh encodes the regulatory light chain of Myosin II, which normally accumulates at the furrow front. Likewise, in sqh-gfp embryos, functional myosin II-GFP (Myo-GFP) reveals the furrow front. Injections of the anti-Lva antibody cause severe furrowing defects. At ~50 min into cycle 14, furrow progression is clearly inhibited near the site of injection, where the antibody is most concentrated. Further from the injection site, furrowing occurs at a reduced rate. Approximately 60 min into cellularization, the embryo's surface near the site of injection begins to dimple, becoming pronounced 10 min later. This effect is presumably due to the pulling force exerted by the contractile apparatus on the surface of the embryo where furrowing has failed. To directly examine the effects of anti-Lva antibodies on Golgi, some injected embryos were fixed and analyzed by immunofluorescence using the anti-p120 cis-Golgi antibody. In embryos injected with the concentrated anti-Lva antibody, the normal punctate distribution of p120 is not observed and instead p120 is found throughout the apical cytoplasm. When the concentrated anti-Lva antibody is Cy5 tagged, the Lva protein can be seen to disperse within minutes of the injection, and the residual Lva protein left associated with some Golgi show the Golgi broken down and sessile (Sisson, 2000).

Furrow progression is also inhibited upon injecting monovalent anti-Lva Fab antibody. The relatively uniform effect of the anti-Lva Fab antibody on furrow progression appears to result from the free diffusion of the antibody within the embryo, in contrast to the IgG, which remains concentrated near the site of injection (Sisson, 2000).

The inhibition of furrow progression resulting from anti-Lva antibody injections suggest that Golgi-derived membrane vesicle export might be required for furrow progression. This predicts that the fungal toxin brefeldin A (BFA), a potent inhibitor of Golgi-derived membrane vesicle transport, should inhibit furrow progression. Injection of BFA severely inhibits furrow progression. Although the Myo-GFP at the furrow front appears discontinuous during the slow phase, furrow progression is relatively normal; however, the fast phase is absent. By 60 min, the furrows in BFA-injected embryos have invaginated only 50% as far as those in the control embryo. When BFA is injected later, at ~25 min into cycle 14, furrow progression is only mildly affected. Because BFA is a small, hydrophobic molecule, it diffuses rapidly through the embryo, resulting in a uniform effect (Sisson, 2000).

Lva lacks significant overall sequence similarity to other proteins; however, it does resemble members of a growing class of proteins called golgins (Chan, 1998 and Warren, 1998). Although golgins are themselves diverse with respect to size and sequence, they all share two common protein features: each is predicted to form an extensive coiled-coil and associate with Golgi membrane. These are also defining features of Lva. Although the precise function of the golgins is unknown, recent work indicates that some golgins form a filamentous matrix between Golgi compartments, and Golgi compartments and COP I vesicles that may facilitate membrane budding and fusion, and maintain Golgi structural integrity (Warren, 1998; Waters, 1999; Sisson, 2000).

Two models have been proposed for how a special isoform of mammalian ß-Spectrin, ßIsigma-Spectrin, might function on Golgi. Its principle role might be to specifically facilitate vesicle formation and/or transport by associating with adaptor complexes like COP I on donor membrane. Alternatively, it may form a dynamic scaffold over the Golgi's surface, which would provide structural integrity and facilitate vesicle transport and cytoskeletal interactions. Because both golgins and Golgi-Spectrin may play structural roles, it has been suggested that they might interact and participate in some common Golgi functions (De Matteis, 1998). Consistent with this proposal, it has been established that Lva and Spectrins stably associate. In addition, upon injecting a specific anti-Lva antibody into cellularizing embryos, a dramatic redistribution of the cis-Golgi marker p120 occurs, MT-based Golgi movement is blocked, and furrow progression is severely inhibited. The most parsimonious explanation for these effects is that Lva forms part of a Golgi-based scaffold that supports MT interactions and membrane transport, including membrane vesicle export required for furrow progression. Interestingly, the distribution of p120 in embryos injected with the anti-Lva antibody closely resembles the normal distribution of the resident ER chaperone BiP, suggesting that perhaps inhibition of Lva function blocks ER to Golgi membrane transport. Because Lva Golgi associates in Drosophila S2 cultured cells and in all embryonic cells examined, it is suspected Lva serves an essential Golgi function (Sisson, 2000).

Lva and Spectrin might recruit MT-binding proteins to the surface of Golgi. Based on the rate (~0.1-0.2 µm/s) and direction of Golgi movement, it is possible that cytoplasmic dynein or a minus-end-directed kinesin are responsible. Interestingly, the mammalian dynactin complex has recently been shown to associate with Golgi via Spectrin. In addition, CLIP170, the human homolog of CLIP190, has been found to specifically recruit dynactin to the plus-ends of MTs. Although dynein and p150^Glued do not appear to form a stable association with Spectrins in the experiments described here, a small proportion of CLIP190 and Lva stably associate and colocalize to Golgi bodies. These observations raise the possibility that CLIP190 and the dynactin complex may participate in linking Golgi to MTs during cellularization through Golgi-Spectrin or perhaps a transient association with the Lva/Spectrin complex that was not detected by co-IP experiments (Sisson, 2000).

Interestingly, the final wave of MT-dependent Golgi movement that is observed from the furrow front to the apical cytoplasm perfectly coincides with a shift in the position of plasma membrane (PM) junctions from the basal furrow front to the apical furrow wall. Because polarized secretion is required for apical/basal cell polarity in mammalian epithelia, it is possible that the apical Golgi movements control the dynamic shift in the position of junctions during cellularization in Drosophila. The final wave of Golgi movement may also represent an efficient strategy for gathering Golgi into forming cells before the end of cellularization (Sisson, 2000).

By using fixation methods optimized for the preservation of both MTs and F-actin, it has been found that alpha-Spectrin associates with Golgi bodies in blastoderm embryos. This is the first demonstration of a Golgi-associated Spectrin in invertebrate cells, and the first instance of Golgi-associated alpha-Spectrin in any cell. These findings suggest that while mammalian cells have a Golgi-specific isoform of ß-Spectrin, Drosophila blastoderms appear to distribute the same alpha-Spectrin to both the PM and Golgi. Whether a special posttranslational modification of alpha-Spectrin confers its Golgi localization is unknown. However, PM- and Golgi-Spectrin are distinctly sensitive to immunofluorescent preparations, indicating that they may possess some compositional differences; certainly, the association between Golgi-Spectrin and Lva is apparently one difference. The sensitivity of Golgi-Spectrin to these preparative conditions may explain why Golgi-Spectrin has not been previously described in Drosophila, and neither ß- or ß_H-Spectrin is detected on Golgi (Sisson, 2000).

The source of membrane for cellularization has been controversial. One idea is that PM microprojections (MPs) observed above nuclei before furrow formation flatten to give rise to the furrow. The MPs could represent pre-existing PM that is thrown into patches of villi by cortical contraction, and/or they may result from de novo intracellular membrane delivery early during cellularization. In either case, estimates suggest the MPs can only account for one half of the total membrane required for furrowing; thus, the remaining membrane must come from intracellular stores, and numerous sources have been proposed (Sisson, 2000 and references therein).

Together, several observations indicate that Golgi-derived membrane export is essential for cellularization. First, the association between Golgi bodies and the furrow front in live embryos suggests the Golgi may provide membrane to the furrow front through short-range vesicle delivery, consistent with EM data. In addition, the severity of the furrowing defects induced by the anti-Lva IgG and Fab fragments and BFA suggests that Golgi bodies are a significant source, and perhaps the principle source, of membrane for furrow progression (Sisson, 2000 and references therein).

Although both the slow and fast phases of furrow progression appear to depend on Lva function, the fast phase appears to be particularly sensitive to BFA. This may reflect a difference in the specific mechanism of exocytosis for each phase of furrow progression. For example, furrow progression during the slow phase, which precedes the fast phase, may simply rely on pre-existing Golgi membrane stores, while the fast phase depends on BFA-sensitive active transport from the ER to Golgi. The timing of the BFA sensitivity is consistent with the proposal that membrane required for the fast phase is actually exported to the cell surface early during cycle 14 and 'stored' in the form of MPs until the onset of the fast phase, when it is used to complete furrow progression (Sisson, 2000 and references therein).

For over two decades, the generally accepted view of animal cell cytokinesis has been that an actomyosin-based contractile ring provides annular force, which, like a 'purse-string,' pulls the plasma membrane inward to form a cleavage furrow. However, evidence presented here, in addition to other recent studies showing that Syntaxin and phosphatidylinositol 4-kinase (Brill, 2000) are required for cytokinesis, indicates that the mechanism of animal cell cytokinesis is more complex and requires targeted membrane secretion. The results of the characterization of Lava lamp extend these previous observations by directly showing that active Golgi-derived membrane vesicle secretion is essential for cytokinesis. Therefore, a more accurate paradigm for animal cell cytokinesis includes both actomyosin-based contraction and membrane vesicle secretion. Although this revised model does not diminish the importance of the contractile ring, it does raise an important question concerning its precise function. Namely, does the contractile ring provide a driving force for cytokinesis, as commonly thought, or an elastic force, which accommodates and guides new plasma membrane in the formation of a cleavage furrow? This newer perspective also leads to changes in the way the functions of proteins already known to be required for cytokinesis are viewed. For instance, the Septins associate with the contractile ring and have been proposed to recruit and/or stabilize the contractile ring. However, recently the mammalian Septins have been shown to physically associate with the Sec6/8 complex and Syntaxin, proteins implicated in polarized secretion and membrane vesicle fusion, respectively. So, perhaps the role of the Septin complex during cytokinesis is to facilitate the proper docking and/or fusion of membrane vesicles with the cleavage furrow. These and other considerations will undoubtedly become the subject of future work. The current study has laid a basic foundation for future investigations in Drosophila, and will hopefully lead to a better understanding of how contraction and membrane secretion combine to bring about cytokinesis and other dramatic changes in cell shape (Sisson, 2000 and references therein).

GENE STRUCTURE

Bases in 5' UTR - 842

Exons - 9

Bases in 3' UTR - 334

PROTEIN STRUCTURE

Amino Acids - 2798

Structural Domains

The Lva gene is predicted to encode an acidic (pI 4.6) protein of 2,798 amino acids with a predicted molecular weight of 318 kD. Amino acid sequence analysis with COILS indicates that Lva is likely to form an extended coiled-coil. Many rod-shaped proteins migrate slower than expected by SDS-PAGE, which may explain the discrepancy between the predicted and apparent molecular weights for Lva. Lva searches have not revealed homologs of Lva; however, BLAST does share features in common with golgins, a class of Golgi-associated proteins (Sisson, 2000).

date revised: 10 February 2001

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