InteractiveFly: GeneBrief

combover: Biological Overview | References

Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster, individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. This study shows that combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes (Steinhauer, 2019).

Proper differentiation of germline cells into eggs and sperm is essential for the perpetuation of a species. Sperm cells in mammals and Drosophila melanogaster develop from germline stem cells that produce mitotic spermatogonia, which ultimately undergo meiosis and terminal spermatid differentiation. Drosophila has long been a powerful model organism for studying spermiogenesis, which begins after meiosis, when the syncytial spermatids derived from a single gonialblast (the differentiating stem cell daughter) begin morphological changes required for their differentiation, including mitochondrial differentiation, flagellar elongation, nuclear compaction and acrosome formation. In Drosophila, after meiosis is completed, mitochondria aggregate around the basal body on one side of the nucleus. Subsequently, the mitochondria fuse to form the 'Nebenkern'. As differentiation proceeds, spermatids form acrosomes, their nuclei remodel and compact, and sperm tails (flagella) elongate. During spermatid elongation, both axonemal microtubules (MTs) and the mitochondrial Nebenkern extend to form the flagellum, composed of a central axoneme flanked by major and minor mitochondrial derivatives. Drosophila sperm axonemes are structurally similar to other axonemes, containing a central MT pair ringed by nine outer MT doublets (Steinhauer, 2019).

In Drosophila, as in mammals, incomplete cytokinesis during sperm development leads to cytoplasmic sharing between sister spermatids. Following terminal differentiation, the inter-spermatid cytoplasmic bridges are dissolved and the spermatids' cytoplasmic contents are removed. This process in Drosophila, called individualization, is carried out by the actin-rich individualization complex (IC) that first forms adjacent to the needle-shaped spermatid nuclei, which by this point are clustered in the basal testis. The IC is composed of 64 actin cones, one for each spermatid nucleus of the germline cyst, and it shares similarities with actin comets found on endocytic vesicles. As individualization proceeds, the actin cones of the IC move synchronously away from the nuclei toward the apical domain of the testis, traversing the spermatid tails until they reach the end of the flagella. Each moving IC generates a growing cystic bulge that contains the extruded cytoplasmic contents of the individualizing cyst. When the IC and associated cystic bulge reach the end of the flagella, they form a waste bag (WB) full of discarded organelles, degradation of which may occur by an apoptosis-like program. As a result of individualization, each streamlined spermatozoon resides within its own plasma membrane, most of its cytoplasm has been extruded, and it no longer maintains connections to its sister spermatids. Once this process has been completed, mature sperm are coiled in preparation for release into the seminal vesicle, and WBs and abnormal sperm are eliminated within the base of the testis (Steinhauer, 2019).

Elegant prior studies have provided a detailed view of the polarized IC structure. At the leading edge of the actin cones, F-actin filaments form a meshwork under the control of the Arp2/3 actin-nucleating complex, whereas at their rear, F-actin is organized into parallel bundles by the activity of the actin-bundling proteins Quail/Villin, Chickadee/Profilin and Singed/Fascin. IC activities are thought to be segregated, with the front meshwork being responsible for cytoplasmic extrusion and the rear bundles responsible for movement along the sperm tails. During individualization, cones accumulate actin, and actin polymerization, but not myosin motor activity, is essential for IC progression (Steinhauer, 2019).

Previously identified combover (cmb), a gene encoding two major protein isoforms that are predicted to be largely intrinsically disordered and lack any known domains, as a Rho kinase substrate that acts as a planar polarity effector (PPE) to affect Drosophila wing hair (trichome) formation (Fagan, 2014). During pupal development, each cell of the fly wing forms an actin-rich hair, surrounded by membrane, that points towards the distal tip of the wing. Polarized wing hair formation is governed by the non-canonical Wnt-planar cell polarity (PCP) pathway, during which core PCP proteins including Frizzled (Fz), Dishevelled (Dsh), Van Gogh (Vang), Prickle (Pk), Diego (Dgo), and Flamingo (Starry night) localize asymmetrically across proximal-distal wing cell borders. PCP-generated asymmetry leads to proximal enrichment of the PPE proteins Inturned, Fuzzy, Fritz and Multiple wing hairs (Mwh), which prevent ectopic wing hair formation. Additionally, Mwh prevents the formation of multiple prehairs, and it bundles actin to restrict hair formation to a single hair during trichome outgrowth. Although cmb mutants show no wing phenotype, Cmb overexpression causes formation of a multiple hair cell (MHC) phenotype that is enhanced by removal of one gene dose of Rok or the PPE genes, including mwh (Fagan, 2014). Indeed, the two major isoforms of Cmb, PA and PB, physically interact with Mwh, and the MHC phenotype observed in mwh single mutants is partially suppressed in mwh cmb double mutants. It was therefore suggested that Cmb promotes actin-based wing hair formation under control of Rok, a function that is antagonized by Mwh (Steinhauer, 2019).

It was noticed that cmb mutants are male sterile. This study found that this is due to a requirement for cmb in the germline, in the actin-based process of spermatid individualization. Detailed histochemical and electron microscopic analyses of cmb mutant testes show that early cyst formation is normal. Furthermore, ICs form normally and start to move but do not remain properly aligned, and, although they initially recruit actin at normal levels, they fail to maintain and accumulate actin during IC progression. Despite evidence that Cmb affects actin-related processes (wing hair formation in the pupa and individualization of sperm), Cmb does not directly interact with actin in co-immunoprecipitation (CoIP) or co-sedimentation assays. Furthermore, it was shown that the role of cmb during individualization is independent of and distinct from PCP genes. Significantly, Cmb interacts with the axonemal component Radial spoke protein 3 (Rsp3/CG32392), knockdown of which causes individualization phenotypes highly similar to those of cmb mutants, suggesting that Cmb coordinates IC movement with the axoneme during biogenesis of functional sperm (Steinhauer, 2019).

Combover/CG10732, a novel PCP effector for Drosophila wing hair formation

The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, Combover (Cmb)/CG10732 was identified as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. These data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh (Fagan, 2014).

Rho kinase, a member of the AGC kinase family which also includes PKC and Akt was originally identified as a RhoA effector reorganizing the cytoskeleton by promoting the formation of actin stress fibers. In Drosophila, Rok was shown to act downstream of Fz and Dsh in the non-canonical Wnt/Planar Cell Polarity pathway causing ommatidial rotation and structural defects in the eye and multiple hairs cells in the wing. This study has identified Combover/CG10732 as a novel substrate of Rok. A cmb protein null allele lacking both Cmb protein isoforms that is homozygous viable. Homozygous cmb mutants display no visible phenotype in the wing or in sections of the adult eyes. As a reduction or an excess of actin polymerization can cause MHCs, this study assessed the overexpression phenotype of Cmb. Indeed, overexpression of either Cmb isoform caused a multiple hair cell phenotype that is strongly dominantly enhanced by rok and the fy/in/mwh PCP effectors, validating the in vitro screening approach to identify PCP effectors. Importantly, the cmb mutation suppresses the MHC phenotype of mwh in double mutants. The data thus indicate that Cmb, while not essential for wing hair formation, nevertheless promotes trichome formation in vivo (Fagan, 2014).

It has been noted that known phosphorylation sites of Rok targets such as ERM proteins, Vimentin, Myosin regulatory light chain, or Adducin, often follow the consensus site [R/K]XX[S/T] or [R/K]X[S/T]. Of the five Rok sites identified in in vitro kinase assays followed by MS analysis, only S300 is preceded by a basic residue at position [-2] (RT[S]). In all other cases, no basic amino acid is found at position [-1] or [-2]. However, T46, T206, T368, and T370 are all followed by a Proline, more typical of MAP kinase phosphorylation sites. Nevertheless, mutation of these sites strongly reduced Cmb phosphorylation in vitro (Fagan, 2014).

In rok mutants, multiple hairs form at the distal end of wing cells. Similarly, overexpression of either Cmb isoform causes MHCs that originate at the distal end of cells, distinct from the in/fy group of PCP effectors and mwh, which form MHCs around the periphery of the cells (note that in mwh mutants, actin patches are initially even formed all over the apical cell surface). Importantly, reduction of rok activity by the removal of one gene dose (by two different alleles or a deficiency) increases the number of MHCs, suggesting an inhibitory effect of Rok on Cmb. It was suggested that Myosin II, which is concentrated at the site of prehair initiation and whose activity is regulated by Rok via phosphorylation of its regulatory light chain (MRLC), must be within an optimal range to properly bundle actin and to ensure the formation of a single hair. Consistent with the genetic interaction between cmb and rok, it is possible that in addition to regulating MRLC, Rok might also inhibit a potential hair promoting activity of Cmb, although it cannot be excluded that Rok/MRLC activity acts in parallel to the effect Cmb exerts on wing hair formation (Fagan, 2014).

mwh and the in/fy group of PCP effectors all have been implicated in restricting actin hair initiation to the distal vertex of the cells by inhibiting proximal hair assembly. Core PCP signaling ensures proper proximal localization of In, Frtz, and Fy proteins (and thus inhibition of prehair formation) to the proximal end of wing cells leading to the formation of a single wing hair on the distal side (Fagan, 2014).

cmb-RA or cmb-RB cause the formation of MHC phenotypes upon overexpression with several wing drivers. Importantly, this overexpression phenotype is enhanced by the removal of one gene dosage of the PCP effectors fy, frtz, in, and mwh as well as deficiencies uncovering those loci. These genetic interactions suggest cmb could exert a positive effect on hair initiation, although such a function would play a supportive or redundant role as neither a lack or ectopic trichomes are found in cmb mutants (Fagan, 2014).

Significantly, this study showed that Cmb physically interacts with Mwh in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, while no vertebrate homologs of cmb have been identified, orthologs of both cmb and mwh were found outside of the insects in the genomes of the crustacean Daphnia magna and of the hard-bodied tick Ixodes scapularis. Ixodes is a member of the Chelicerata, the most basally-branching euarthropod clade that split from the remaining arthropod groups in the Cambrian. The presence of mwh and cmb in Ixodes may be indicative of an ancient protein-protein interaction that has been retained throughout arthropod evolution. Because both Ixodes and Daphnia lack wings, the Mwh/Cmb interaction likely performed different, possibly additional function in the ancestral arthropod. Consistent with this, mwh mutants cause other cuticular hair defects in other regions of the Drosophila body. Alternatively, the Mwh/Cmb interaction evolved much later than the appearance of both of these genes in the genome of the ancestral arthropod. The roles of and interactions between Cmb and Mwh proteins in more non-insect arthropods needs to be further explored (Fagan, 2014).

The presence of both mwh and cmb orthologs in the genomes of members of all holometabolous insect orders may indicate that the Mwh and Cmb interaction is also conserved in this insect clade. The retention of these two genes in members of the more basally-branching hemipteran orders, however, is less conserved. The conservation of mwh and cmb in Holometabolata may be due to their shared mode of wing development, i.e. via internal wing imaginal discs. This is in contrast to the mode of wing development in hemimetabolous insects by which the wings develop as buds outside of the body. Further study into the association of wing development and Mwh/Cmb interactions in other insect orders is needed to elucidate these findings (Fagan, 2014).

Interestingly, PCP effector mutations generally enhance each other. For example, the hypomorphic frtz3 allele is enhanced by weak alleles of in or fy in double mutants. Analogously, removal of a gene dosage of mwh in a fy or in background, enhances their MHC phenotype. In contrast, the MHC phenotype of mwh mutants was (partially) suppressed in mwh cmb double mutants, as significantly fewer cells formed additional hairs. This interaction is likely specific, because this study found it with the temperature sensitive mwh6 allele and with the spontaneous mwh1 allele, two alleles of independent origin unlikely to carry a similar second site mutation and thus further supporting the physiological function of Cmb as a PCP effector. cmb is the only gene reported so far to suppress mwh. Importantly, as mwh1 and cmb both are null alleles (cmbKO lacks expression of both protein isoforms), this result suggests that Mwh acts upstream of and normally antagonizes Cmb and that the derepression of Cmb thus may contribute to the MHC phenotype of mwh mutants (Fagan, 2014).

Unfortunately, tg Cmb antibodies do not detect endogenous Cmb protein in the developing pupal wing. Nevertheless, Cmb expressed in the posterior compartment of the wing under the control of en-Gal4 localizes apically in a punctate pattern. In cells that appear to express at a lower level, Cmb is enriched at the circumference of the cells, but shows no proximo-distal enrichment. Although it cannot be excluded that Cmb localization is an overexpression artifact, this appears unlikely, because it would be expected that Cmb would fill the cells rather than to localize specifically apically. Importantly, Cmb likely localizes to the area of wing cells where Mwh is present, as Mwh known to be initially enriched apically towards the proximal side, further supporting the model that a positive effect of Cmb as a novel PCP effector on wing hair formation may be restricted by Mwh (Fagan, 2014).

Search PubMed for articles about Drosophila Combover

Fagan, J. K., Dollar, G., Lu, Q., Barnett, A., Pechuan Jorge, J., Schlosser, A., Pfleger, C., Adler, P. and Jenny, A. (2014). Combover/CG10732, a novel PCP effector for Drosophila wing hair formation. PLoS One 9(9): e107311. PubMed ID: 25207969

Steinhauer, J., Statman, B., Fagan, J. K., Borck, J., Surabhi, S., Yarikipati, P., Edelman, D. and Jenny, A. (2019). Combover interacts with the axonemal component Rsp3 and is required for Drosophila sperm individualization. Development 146(17). PubMed ID: 31391193

date revised: 5 November 2019

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