Journal of Cell Science 107, 1863-1873 (1994) 1863 Printed in Great Britain © The Company of Biologists Limited 1994 The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos Marya A. Postner and Eric F. Wieschaus* Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA *Author for correspondence SUMMARY After the 13th nuclear division cycle of Drosophila embryo- actin caps and within metaphase furrows. In cellularizing genesis, cortical microfilaments are reorganized into a embryos, nullo co-localizes with the actin-myosin network hexagonal network that drives the subsequent cellulariza- and invaginates along with the leading edge of the plasma tion of the syncytial embryo. Zygotic transcription of the membrane. The serendipity-α (sry-α) protein co-localizes nullo and serendipity-α genes is required for normal struc- with nullo protein to the hexagonal network but, unlike the turing of the microfilament network. When either gene is nullo protein, it localizes to the sides rather than the deleted, the network assumes an irregular configuration vertices of each hexagon. Mutant embryos demonstrate leading to the formation of multinuceate cells. To investi- that neither protein translationally regulates the other, but gate the role of these genes during cellularization, we have the localization of the sry-α protein to the hexagonal made monoclonal antibodies to both proteins. The nullo network is dependent upon nullo. protein is present from cycle 13 through the end of cellu- larization. During cycle 13, it localizes between interphase Key words: Drosophila embryo, cytokinesis, contractile ring INTRODUCTION taposition: the sides of each polygon in the array are formed by converting the ‘fuzzy’ actin organization at the cap margins In Drosophila embryos, the early nuclear divisions are not into more finely aligned actin filaments (Simpson and followed by cytokinesis and the embryo initially develops as a Wieschaus, 1990). Each hexagonal interface of the actin syncytium. This organization persists until after the 13th network defines the site of membrane invagination. Cytoplas- division, at which time the embryo consists of approximately mic myosin co-localizes with actin in the hexagonal network 6,000 nuclei arranged in a monolayer in the embryo’s cortex. (Warn et al., 1980; Young et al., 1991), and both filamentous Subdivision of the cortical cytoplasm into individual cells is actin and functional cytoplasmic myosin are required for known as cellularization. During this process, plasma membrane invagination (Zalokar and Erk, 1976; Foe and membrane invaginates from the surface in a roughly hexagonal Alberts, 1983; Kiehart et al., 1990). Contraction of this pattern, precisely separating each nucleus from its immediate actin/myosin array has been postulated to provide a mechanis- neighbors. Once the membrane has reached a depth of about tic force driving the invagination. This role for the actin- 25 µm, the base of the invaginating membrane furrow begins myosin network is based in part on an analogy with the ‘con- to widen, eventually separating the newly formed cells from tractile rings’ of actin and myosin that are thought to drive the underlying yolk. The resulting cellular blastoderm consists invagination of the plasma membrane during conventional of a single layer of columnar cells surrounding the central yolk cytokinesis (Mabuchi, 1986; Salmon, 1989; Schroeder, 1990; sac. Satterwhite and Pollard, 1992). During the first ten minutes of cycle 14, a highly organized Most of the components of the hexagonal array are supplied array of F-actin is formed on the cytoplasmic face of the by maternal transcription during oogenesis and are already invaginating plasma membrane (Fig. 1, see also Warn and present as RNA or protein in the unfertilized egg. Cellulariza- Magrath, 1983; Simpson and Wieschaus, 1990; Warn and tion, however, marks the point in Drosophila development Robert-Nicoud, 1990; Schejter and Wieschaus, 1993). Prior to when the embryo becomes dependent on gene products formation of the array, the cortical actin of the embryo is supplied by the embryo’s own transcription (Arking and organized in ‘caps’ overlying each nucleus. Initially the caps Parente, 1980; Edgar and Schubiger, 1986). A small number of formed in cycle 14 resemble those seen in the preceding inter- genes have been identified whose zygotic products are required phases. However, in contrast to earlier caps, which remain for the formation of a normal actin array (Wieschaus and static during interphase, the cycle 14 caps soon enlarge until Sweeton, 1988; Merrill et al., 1988). Embryos lacking either the their bases touch. The actin array arises in regions of cap jux- nullo or the serendipity-alpha (sry-α) gene show very similar 1864 M. A. Postner and E. F. Wieschaus abnormalities in the actin array: some of the sides of the cies that uncover the nullo locus. The deficiencies Df(1)6F1-2 and hexagons are unusually thick while others are extremely thin or Df(1)LIMDF were most commonly used (for description see Simpson missing altogether (Fig. 1B,C). In nullo embryos, the initial few and Wieschaus, 1990; Rose and Wieschaus, 1992). Embryos with the minutes of network formation appear normal (Wieschaus and sry-α phenotype were collected from a stock that is heterozygous for α Sweeton, 1988; Simpson and Wieschaus, 1990). However, at Df(3R)X3F, which uncovers the sry- gene. Since no point mutations the onset of membrane invagination, network formation is exist for either gene, the mutant phenotype only arises in deficiency embryos. The terms ‘nullo mutant’ and ‘sry-α mutant’ are used to incomplete and the uneven distributions and sporadic disrup- describe the deficiency embryos. tions in the actin-myosin network become obvious. Once underway, network and membrane invagination appear to Production and screening of monoclonal antibodies proceed normally: neither the kinetics of membrane extension Monoclonal antibodies to the nullo protein were generated using a nor the length of the newly formed cells is significantly different nullo-glutathione S-transferase fusion protein as the antigen. An in- from that observed in wild-type embryos. However, cleavage frame fusion of the entire nullo protein to the carboxyl terminus of furrows do not invaginate where the network is discontinuous glutathione S-transferase (Smith and Johnson, 1988) was constructed and multinucleate cells form. The only obvious difference in the following manner. The nullo coding region was PCR amplified between nullo and sry-α mutant embryos is that sry-α embryos from the nullo M1 cDNA (Rose and Wieschaus, 1992) using primers have fewer multinucleate cells (Merrill et al., 1988; homologous to the ends of the coding region. The primers also contained an external stretch of bases that lacked homology to nullo Schweisguth et al., 1990; E. Schejter, personal communication). α and contained an EcoRI restriction site. The resulting PCR product Molecular characterization of the nullo and sry- genes has was digested with EcoRI and ligated into the EcoRI site of pGEX-2T revealed that both genes encode single, blastoderm-specific (Pharmacia). The orientation of the inserts was determined by restric- transcripts that are uniformly distributed throughout the tion mapping. syncytial embryo and accumulate in large amounts over a short The pGEX-2T-nullo plasmid was transformed into Escherichia coli period of time (Vincent et al., 1985; James and Vincent, 1986; strain JM101 and the production of fusion protein was induced with Rose and Wieschaus, 1992). Transcript levels reach a sharp 1 mM IPTG (Pharmacia). After 3 hours, the cells were harvested. peak around the onset of cellularization and subsequently Because the fusion protein was stubbornly insoluble, it was excised decrease in a rapid, spatially patterned manner. The main dif- from a preparative acrylamide gel, electroeluted with Elutrap ference in the transcription pattern of the two genes is that sry- (Schleicher and Schuell), and dialyzed against MTPBS (Smith and α Johnson, 1988). The purified protein was used to immunize two mice transcripts arise, peak and decline slightly later than nullo. and monoclonal antibodies were produced following standard Neither gene is required for the transcription of the other (Rose protocols (Harlow and Lane, 1988). Supernatants from the monoclonal and Wieschaus, 1992). lines were tested by western blot for recognition of the fusion protein The sry-α protein is 58 kDa in size, lacks extensive and of glutathione S-transferase. The 23 lines that recognized only the homology to any known proteins and shows few structural fusion protein were tested by western blot for reactivity with proteins motifs (Ibnsouda et al., 1993). Immunolocalization indicated from two- to three-hour Ore-R embryos. Monoclonal supernatants that during cellularization sry-α protein localizes to the leading 5C3-12 and 2F8-18 specifically recognize the nullo proteins. Because edge of the invaginating plasma membrane (Schweisguth et al., the 5C3-12 antibody reacts more strongly with the nullo proteins than 1990). Like its transcript, the sry-α protein is short-lived. The does 2F8-18, it was used preferentially unless otherwise indicated. Monoclonal antibodies to the sry-α protein were generated using a nullo gene is predicted to encode a 23 kDa protein lacking α α truncated version of the sry- protein as the antigen. This protein, homology to known proteins, including the sry- protein. which contains amino acids 46 to 530 of sry-α, was produced from a Sequence analysis demonstrated that the nullo protein has an T7 RNA polymerase-inducible vector (Studier and Moffat, 1986). The excess of basic amino acids (predicted pI is 11.4) and plasmid, pPαNN, contains a NarI to NcoI fragment of the sry-α gene suggested that the protein may be myristoylated (Rose and cloned in pET3a. It was generously provided by Alain Vincent. The Wieschaus, 1992). However, previous studies did not address plasmid was transformed into E. coli strain BL21-Lys S. Fusion intracellular localization of the nullo protein or its specific cell protein production was induced for three hours with 1 mM IPTG.