Cellularization of the Drosophila embryo is a specialized form of cytokinesis that couples membrane growth with the formation of a polarized epithelium. slow as molasses (slam) is essential for polarized growth of the plasma membrane during cellularization. In slam mutant embryos, the furrow canal is disorganized, and polarized insertion of transmembrane proteins is disrupted. slam shows a striking developmental induction during the slow phase of cellularization, and Slam protein localizes to the furrow canal and the basal junction. Slam colocalizes with the junctional proteins Armadillo/ß-catenin, the PDZ domain-containing protein Dlt (renamed Patj), and Myosin (see Zipper) and is also required for their proper membrane localization. These results suggest that developmental induction of Slam organizes the polarized growth of membrane via the recruitment of membrane-targeting proteins at adherens junctions (Lecuit, 2002 and Stein, 2002).

slam is also required for germ cell migration. In slam zygotic mutants, germ cells fail to transit off the midgut into the mesoderm. slam is required at this stage in parallel to HMG Coenzyme A reductase, another germ cell migration gene. Because slam RNA and protein are expressed earlier than the time when defects are observed in germ cell migration, it is proposed that Slam is required for the localization of a signal to the basal side of blastoderm cells that is needed later in the posterior midgut to guide germ cells (Stein, 2002).

Cleavage of the Drosophila syncytial embryo is known as cellularization and provides a striking example in which morphogenetic processes are associated with profound rearrangements of the plasma membrane. During cellularization, the plasma membrane surface grows about 30-fold, invaginates between cortical nuclei, and produces a polarized epithelium. The first 13 nuclear divisions occur in a single cytoplasm and result in about 5000 nuclei located at the cortex of the embryo. At the beginning of cycle 14, the divisions cease, and the membrane invaginates between each nucleus, partitioning them into single unit cells. The first 35-40 min of cellularization (slow phase) account for the first 10 μmmeters of new membrane along what will become the basal-lateral surface of each cell. The speed of invagination rapidly increases during the last 15-20 min (fast phase) and stops when the invaginating front is located about 30 μmeters inside the embryo. By using pulse labeling of the plasma membrane, it has been shown that membrane growth during cellularization involves the regulated mobilization of membrane pools from the secretory pathway to precisely defined sites (Lecuit, 2000). The modes of membrane growth are different in slow phase and fast phase, paralleling the change in the rate of membrane invagination. During slow phase, membrane growth is concentrated apically. When the plasma membrane is labeled with a fluorescent lectin before membrane invagination begins, the apical label is rapidly lost, whereas it persists and even accumulates in the region that will form the front of invagination called furrow canal (FC). The same pattern of membrane dynamics is maintained as the embryo progresses through the end of slow phase; apical membrane is rapidly replaced with intracellular unlabeled membrane, whereas the basal-lateral membrane remains labeled as it forms and grows. This observation suggests that the apical membrane is the principle site of new unlabeled membrane insertion and that, during cellularization, a lateral domain with different membrane turnover properties is established adjacent to the original apical surface. This lateral domain is apparent prior to invagination and persists during slow phase. During fast phase, a new site of membrane insertion is added in an apical-lateral region of the plasma membrane (Lecuit, 2002).

In order to understand the mechanisms underlying the regulated membrane addition during cellularization, a search was carried out for genes required for stage-specific membrane growth. Drosophila cellularization occurs at a key developmental transition similar to the mid blastula transition in Xenopus (MBT) and is characterized by the induction of zygotic gene expression. Two categories of gene products contribute to membrane invagination during cellularization: maternally supplied RNAs and proteins deposited by the mother during oogenesis and zygotic gene products induced during cellularization. Genetic screens have identified a surprisingly low number of genomic regions required zygotically for cellularization, suggesting that less than ten nonredundant genes might control the entire process (Merrill, 1988; Wieschaus, 1988). This finding suggests that such zygotic genes might correspond to key regulators whose expression at specific points during cellularization activates a maternal machine poised to deliver membrane pools to specific sites of the plasma membrane (Lecuit, 2002).

A new gene, slow as molasses, has been identified that is required for the growth of the basal-lateral membrane during cellularization. slam expression is rapidly induced during slow phase at the beginning of cycle 14. In slam mutant embryos, the membrane that would normally form the furrow canal and basal lateral membrane never assumes the smooth morphology devoid of villous projections characteristic of that region. The furrow canal and basal junction do not form. This morphological defect is accompanied by defects in apical membrane trafficking, such that transmembrane proteins like Neurotactin (Nrt) and Toll accumulate abnormally in the apical cytoplasm. slam encodes a novel protein that localizes in a polarized fashion to the furrow canal and to the basal adherens junction but not apically. Slam colocalizes with and is required for the proper membrane accumulation of junctional components such as ß-catenin/Armadillo (Arm) and the PDZ domain protein Patj and of Myosin. Continued expression of slam after cellularization leads to a polarized distribution of the protein in the apical adherens junctions along the anterior and posterior borders of cells. In such embryos, Slam recruits Myosin to the junction in a similar asymmetric pattern, arguing that the two proteins interact in vivo. It is proposed that Slam organizes the polarized growth of the basal-lateral membrane via the regulation of membrane insertion at the level of basal junctions. These findings substantiate the notion that morphogenesis and polarization of the plasma membrane are inherently linked processes (Lecuit, 2002).

Their small number and the specificity of their phenotypes suggest that genes required zygotically for cellularization may define key steps regulating or inducing specific aspects of the process. Previous experiments have identified different patterns of membrane insertion during the slow and fast phases of cellularization. During slow phase, growth of the membrane is concentrated apically. But, during fast phase, a new site of membrane insertion is superimposed apicolaterally. This striking observation suggests that the subdivision of the entire process based on the kinetics of invagination might also reveal distinct mechanisms of membrane insertion and growth. If so, one predicts that genes specifically regulating membrane growth in one phase or the other ought to exist. Several features of slam are consistent with such a specific role. In slam mutant embryos, membrane growth is inhibited during slow phase, while fast phase appears at the right timing. In addition, the induction of slam RNA correlates with that of slow phase. Slam levels peak at the beginning of cycle 14, when slow phase begins. The RNA is then degraded ~40 min later during fast phase. Finally, slam controls the formation of membrane structures that are specific to cellularization: the furrow canal and its associated basal junction as well as the basal-lateral membrane surface. It is likely that Slam expression induces the transition to slow phase and thus controls the onset of cellularization (Lecuit, 2002).

Because slam RNA (and potentially Slam protein) persist when fast phase is initiated, the transition to fast phase may involve the addition or superimposition of a second membrane insertion pathway, such that the observed increase in membrane invagination may reflect the sum of two pathways. This would explain the reduced rate of membrane invagination during fast phase in slam(RNAi) embryos.

slam appears to be unique in the genome in respect to its requirement during slow phase. RNA(i) to slam reproduces the phenotype of embryos deleted for almost the entire left arm of the second chromosome. Translocation screens have identified no other regions that affect membrane growth during the slow phase of cycle 14 (Lecuit, 2002).

slam, however, is not the only gene required zygotically at the beginning of cycle 14: other regions of the genome affect cellularization, although the associated phenotypes are morphologically distinct from those of slam. For example, expression of the nullo gene is required for the stabilization of a basal adhesive junction that isolates the furrow canal and allows the stable accumulation of Myosin (Hunter, 2000). Because slam mutants also show decreased Myosin levels in the furrow canal and fail to form basal junctions, it is possible that both genes work in concert to establish polarized membrane insertion and extension at the level of junctions. Slam may determine the specific character of the protein targeting during slow phase, but the consequences may depend on the existence of specific junctions and other aspects of membrane structure. It is therefore difficult to predict the phenotype of slam expression in stages that do not have the same constellation of factors present at cycle 14. It has not been possible to express Slam earlier than cycle 14 and thus it is not known whether such expression would be sufficient to induce premature cellularization (Lecuit, 2002).

Slow phase is characterized by the formation of two adjacent membrane domains, as revealed by data using pulse labeling of plasma membrane and structural data using electron microscopy. Together, one region of the membrane containing many villous projections has been identified; here lectin label is rapidly removed. In a second adjacent smooth membrane domain, lectin persists and accumulates during slow phase. This early polarity of the plasma membrane is also revealed by the distinct localization of various proteins in the smooth area of the membrane that becomes the furrow canal during slow phase: Dlt (now Patj) and Slam. Note that the same basic pattern persists as invagination, per se, is initiated. The growing lateral membrane is smooth and keeps a high level of labeled membrane in contrast to the apical membrane. Two mechanisms can be envisioned to account for this partitioning of two membrane domains as the basal-lateral membrane grows. (1) As proposed previously (Lecuit, 2000), the newly inserted membrane shows little miscibility with the recipient apical membrane. Such limited mixing has been reported in other systems. Slam's localization in the well organized smooth area of membrane and its exclusion from the area rich in villous projections is consistent with this model. (2) Slam could support a membrane-based scaffold that stabilizes that region and keeps its integrity as new membrane is inserted. The observation that Slam colocalizes with Myosin and the PDZ domain-containing protein Dlt/Patj and that it is required for their proper membrane accumulation lends further support to this view. The induction of Slam could induce the formation of a protein complex that connects the actin-Myosin cytoskeleton to the plasma membrane via Dlt/Patj. PDZ domain-containing proteins such as Dlg/Patj have indeed been implicated in the clustering of transmembrane proteins, such as Fasciclin, and the ion channel Shaker in the synapses (Lecuit, 2002 and references therein).

slam-dependent formation of the furrow canal and of the basal-lateral membrane as a separate membrane domain could alternatively involve an active intracellular membrane transport mechanism. The rapid removal of apically labeled membrane and its accumulation basal laterally could indeed be tightly linked if an endocytic route transfers apical membrane laterally in a manner akin to transcytosis. This transfer of membrane would involve traffic through and sorting from endosomes and intersect the exocytic pathway. Transcytosis is a well-known pathway required for the polarization of hepatocytes, for instance. Slam could stabilize or enhance the junctional insertion of membrane basal laterally and therefore favor the polarized assembly of the lateral membrane. The site of membrane integration and assembly could be the basal junction area, since junctions are known to recruit proteins, such as sec6/sec8, required for the basal-lateral targeting of transport vesicles in MDCK cells and for the associated growth of the basal-lateral membrane. Consistent with this proposal is the finding that the subcellular localization of integral membrane proteins such as Toll and Nrt is abnormal in slam mutant embryos. While these proteins are clearly in part inserted in the apical plasma membrane, they also accumulate in a diffuse apical pattern very distinct from their normal well-defined plasma membrane accumulation. This diffuse cytoplasmic accumulation could not be resolved into punctate structures or well-defined organelles. One possibility is that, in the absence of Slam, the lateral transfer of membrane containing Toll and Nrt is highly inefficient and not polarized at all. Instead of accumulating laterally and contributing to the growth of a distinct membrane domain, vesicles may be routed back to the apical membrane but partially accumulate in the apical cytoplasm. In such a scenario, Slam's unique developmental induction stabilizes the polarized assembly of membrane basal laterally. The suggested interaction between Slam, Myosin, and Dlt/Patj at the level of junctions might be involved in the assembly of membrane-targeting complexes that connect the plasma membrane with the actin cytoskeleton. Biochemical experiments that will identify molecular partners of Slam should be particularly revealing. Although it is not possible to distinguish between these two possibilities, the latter is favored because, unlike the former, it accounts for the observed defects in the localization of Nrt and Toll (Lecuit, 2002).

In conclusion, slam encodes a developmental regulator of membrane morphogenesis during cleavage of the Drosophila embryo. Slam affects the polarized growth of the basal-lateral membrane and its organization as a distinct membrane domain. The data therefore substantiate the notion that membrane growth and polarity are indeed coregulated processes in epithelial cells and that junctions play an important role in this process. Future experiments will reveal whether this also involves a regulation of polarized targeting or not. Slam may prove a very useful entry point into the mechanisms of epithelial polarization and morphogenesis during development (Lecuit, 2002).

GENE STRUCTURE

cDNA clone length - 4093

Bases in 5' UTR - 379

Exons - 3

Bases in 3' UTR - 123

PROTEIN STRUCTURE

Amino Acids - 1196

Structural Domains

A complete slam cDNA identified by the Genome Project comprises an open reading frame 3591 nucleotides long and encoding a large putative 1196 amino acid protein (135 kDa) without any homolog in the database. No signal sequence or putative transmembrane domain is found, arguing that the Slam protein might be cytosolic. BLAST searches fail to identify any conserved protein domain, except for a potential coiled-coil motif between amino acids 516 and 546 and low similarity to protein phosphatases (Lecuit, 2002).

date revised: 5 June 2004

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