BIOLOGICAL OVERVIEW

Chickadee, the Drosophila homolog of Profilin, a protein involved in actin polymerization, was initially characterized in a search for genes that function during cytoplasmic flow from nurse cells to the oocyte. Cytoplasmic flow along actin-based cytoskeletal microfilaments is responsible for transferring cytoplasmic components from nurse cells to oocytes. This process is critical: it ensures that the oocyte is supplied with sufficient stores for initial zygotic development. In chickadee mutants, females are sterile and there is a disruption of the nurse cell cytoplasmic flow and lack of nurse cell cytoplasmic actin networks. Thus chickadee is a perfect candidate to study as a gene involved in actin based dynamics in oocytes (Cooley, 1992)

Chickadee is implicated in several stages of bulk transport (known as 'dumping') of nurse cell contents into the oocyte. In chickadee mutants some nurse cell cytoplasm flows into the oocyte, although the transfer is incomplete. As the microtubule based cytoskeleton is implicated in nurse cell dumping (See beta1 tubulin), it is presumed that both actin based microfilaments and tubulin based microtubules (Theurkauf, 1994) are involved in dumping. What aspect of the actin based cytoskeleton is involved in cytoplasmic dumping? It is likely that sub-cortical actin-based nurse-cell microfilaments play a role in cytoplasmic flow into the oocyte. (There are there are more than three types of cytoskeleton: for example, actin based microfilaments, microtubules, and subcortical microfilaments. See cytoskeleton for a more detailed description of these three). A nonmuscle myosin is found to associate with subcortical actin but not with cytoplasmic networks. These subcortical actin filaments are very sensitive to cytochalasin treatment. Thus, contraction of the subcortical actin could play a role in the bulk movement of nurse cells into the oocyte (Cooley, 1992 and references).

The actin based microfilament cytoskeleton plays an additional role in cytoplasmic dumping. At stage 11, the nurse cells dump their contents into the oocyte through cytoplasmic bridges termed ring canals. Microfilament bundles form in the nurse cells during this process and are apparently required to hold the nurse cell nuclei in place so that they do not obstruct the ring canals and allow rapid flow of nurse cell cytoplasm into the oocyte. It is thought that these cytoplasmic microfilament bundles are non-contractile and serve a structural function (Mahajan-Miklos, 1994). Mutants in chickadee, quail and singed affect actin bundle formation. Profilin, encoded by chickadee, is presumably required for the polymerization of the actin filaments that compose the bundles (Cooley, 1992), while a villin-related protein encoded by quail and a fascin-related protein encoded by singed are thought to be required to cross-link the actin filaments to form the actin bundles. Two components of the actin-lined ring canals have also been identified - an adducin-like protein encoded by hu-li tai shao and a protein containing scruin repeats encoded by kelch (Mahajan-Milos, 1994 and Manseau, 1996 and references).

The actin and tubulin based microfilament components of the cytoskeleton are intimately associated in oocytes; any discussion of one without the other is clearly incomplete. The rapid cytoplamic streaming that occurs during the microfilament-dependent rapid transfer of cytoplasm from nurse cells into the oocytes is dependent on microtubules. This is known since streaming is inhibitable by colcemid, which functions to disrupt microtubules. Mutations in cappuccino and spire repress this microtubule-based ooplasmic streaming. In capu and spir mutants, the bundling of the microtubules at the cortex of the oocyte and streaming of the oocyte cytoplasm occurs prematurely. The effects on capu and spir mutations suggest that these genes are involved in microtubule processes. However, chickadee mutants share the premature streaming phenotype with capu and spir. The mutant phenotype of these three genes is due to a premature bundling of microtubules. Normally microtubules are found at the cortex of the oocyte from stages 8 through 10. In chic and capu mutants, long tubulin-staining fibers are found throughout the oocyte. It is concluded that a protein that interacts with the actin based cytoskeleton, Chickadee, is also involved in maintainence of the tubulin based cytoskeleton. In fact, mutations in chic result in the mislocalization of Staufen, which normally localizes to the posterior pole. Although the phenotype is quite variable, there is a close relationship between the effects of chic on the distribution of microtubules and on the distribution of Staufen (Manseau, 1996).

Therefore, Chickadee, a protein involved in actin cytoskeletal dynamics, is involved in maintainence of the tubulin based cytoskeleton. How can this been? It has been found that Cappuccino interacts directly with profilin. The fact that Capu and Chickadee interact directly, suggests that Capu and Chic may affect the same processes through this interaction. If this is true, then these two proteins may serve as the interface between the actin and tubulin based cytoskeletons (Manseau, 1996).

Another candidate for an protein that interacts with Chicadee may be Diaphanous, which functions in Drosophila to promote cytokinesis, the final separation process that occurs between daughter cells in mitosis. A mammalian protein, p140mDia, has been identified as a downstream effector of Rho, a small GTPase that regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. (For more information see Drosophila Rac1). p140mDia is a mammalian homolog of Diaphanous. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin, the homolog of Chickadee in mammals. The interactions among Rho, Diaphanous and profilin suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes, and that Diaphanous and profilin play a joint role in cytokinesis (Watanabe, 1997).

In Drosophila, Chickadee is involved in proliferation of germ-line cells in both males and females. In the adult female, the germarium is devoid of germline material, resulting in empty follicle cells stacks. Testes of mutant flies contain a few spermatid bundles; testes from older males are markedly smaller than wild type and appear agametic (Verheyen, 1994).

Both macrochaete and microchaete bristles on the head, thorax, legs and wings are affected by chickadee mutation. The bristle shaft is formed as a cytoplasmic extension of the trichogen cell. Its structure is provided by a core of microtubules surrounded by fiber bundles that are in fact actin filament bundles. The ridges seen on the bristle cuticle are formed by cytoplasm of the trichogen cell protruding between the actin filament bundles: therefore these ridges are indicative of the number of bundles formed. chic mutant bristles are thicker and shorter than normal with sharp bends, kinks and forked ends. The ridges on mutant bristles are often thinner, more numerous and disorganized. Mutant bristles contain abundant actin filament bundles, but they are more numerous and somewhat thinner than wild type. Thus the aberrant external ridge morphology correlates with the condition of the underlying actin filament bundles (Verheyen, 1994).

How does profilin/Chickadee function to regulate the actin cytoskeleton? To approach this question one must know something about actin, the substrate of profilin in mammals. Actin is an ATPase that goes through a cycle of nucleotide binding and hydrolysis. ATP hydrolysis is associated with actin polymerization, and ATP-actin polymerizes faster and at a lower critical concentration than ADP-actin. Profilin binding to an actin monomer decreases 1000-fold the affinity of actin for its bound nucleotide. Since ATP is generally present in large excess over ADP, this will have the effect inside cells of replacing bound ADP with ATP. In the absence of profilin, nucleotide exchange on an actin monomer is relative slow. These observations led to a model in which profilin can locally promote actin filament growth (Theriot, 1993).

Profilin functions in another pathway to promote filament growth. It has been shown that the profilin-actin complex adds directly onto the barbed end of growing actin filaments. Since profilin has a relatively low affinity for the barbed end of filaments, the profilin dissociates, leaving the actin filament one subunit longer. It is though that the pathway for barbed-end elongation involving profilin is more thermodynamically favored than the pathway without it; consequently, the final amount of free unpolymerized actin monomer is lower in the presence of profilin than in its absence (Theriot, 1993).

Profilin was originally identified as a component of cell extracts that inhibit actin filament growth in vitro. It was initially assumed that profilin was a major sequestering factor in most cells, and sequestering was considered to be profilin's primary function. However, there is not nearly enough profilin in a typical cell for this, and another protein that occurs in higher abundance (thymosin beta4) may be responsible for monomer sequestration (Theriot, 1993).

It is clear that Chickadee and the actin cytoskeleton play a multifaceted and complex role in Drosophila development. For more information about the role of the actin cytoskeleton in Drosophila morphogenesis, see the Zipper site. Zipper is Drosophila's non-muscle myosin.

GENE STRUCTURE

Two transcripts, of 1.0 and 1.2 kb are present. The complementary DNAs reveal two different 5' exons, suggesting that the two transcripts are products of transcription from alternative promoters. The longer transcript is present in wild-type males and females and also present in RNA isolated from chickadee mutant flies. It is abundant in ovaries but also present in ovarectomized females. The shorter 1.0 kb transcript, however, is virtually ovary specific. It is concluded that the phenotype displayed by female-sterile alleles of chickadee is caused by the absence of the ovary-specific transcript of a gene that is also transcribed from a different promoter (Cooley, 1992). chickadee is immediately adjacent to the eukaryotic initiation factor, eIF4A (Verheyen, 1994).

Exons - Four, with alternative first exons

Bases in 3' UTR - 347

PROTEIN STRUCTURE

Amino Acids - 126

Structural Domains

Drosophila Chickadee is 40% identical to profilins from Saccharomyces cerevisiae, Physarum and Acanthamoeba. The homology increases to greater tha 60% similarity when conserved amino acid substitutions are considered. Profilin from mouse and human is 15 amino acids longer than the protein from lower eukaryotes. Alignment allowing gaps in the proteins shows 25% identity and 50% similarity of the fly protein to profilin of mouse and humans. The homology extends throughout the protein including very high conservation at the carboxy terminus. The region of Acanthamoeba profilin (from amino acids 95-125) has been shown to contain an actin binding site (Cooley, 1992 and references).

date revised: 17 July 97  

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