Gene name - hu-li tai shao
Synonyms - Adducin
Cytological map position-56D3-56D5
Function - signaling
Symbol - hts
FlyBase ID: FBgn0263391
Genetic map position - 2R
Classification - Adducin homolog, N-terminal Head and Neck domains, Adducin Tail, MARCKS domains
Cellular location - cytoplasmic
|Recent literature||McNeill, E. M., Warinner, C., Alkins, S., Taylor, A., Heggeness, H., DeLuca, T. F., Fulga, T. A., Wall, D. P., Griffith, L. C. and Van Vactor, D. (2020). The conserved microRNA miR-34 regulates synaptogenesis via coordination of distinct mechanisms in presynaptic and postsynaptic cells. Nat Commun 11(1): 1092. PubMed ID: 32107390
Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, an unbiased screen was performed for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, miR-34 mutants were discovered to display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.
Drosophila hu-li tai shao (hts; meaning 'too little nursing'), encoding a homolog of mammalian adducin, is required for ring canal formation during Drosophila oogenesis (Yue, 1992). Mammalian adducins comprise a protein family encoded by three genes (α, ß, and γ). The α and γ isoforms (Mr of 103,000 and 84-86,000, respectively) are ubiquitously expressed in human cells; the β isoform (Mr of 97,000) is abundant in erythrocytes and the brain. Subunits assemble into heterodimers, or larger oligomers, through interactions in the globular amino-terminal head domains. In addition, assembly probably occurs through interactions near or within a myristoylated alanine-rich C-kinase substrate (MARCKS) domain by the carboxyl-terminus (Hughes, 1995), which contains PKC phosphorylation sites (Bennett, 1988) as well as binding sites for calcium/calmodulin (Gardner, 1986) and possibly phospholipids. Adducin caps the barbed ends of actin filaments (Kuhlman, 1996; Li, 1998) and promotes their association with spectrin (Gardner, 1987). However, phosphorylation of purified adducin by PKC diminishes its interaction with actin and spectrin (Matsuoka, 1998), whereas addition of exogenous calcium/calmodulin releases adducin from actin (Kuhlman, 1996). Together, these findings indicate that adducin may be involved in exposing actin filament barbed ends in motile cells after activation of certain signal transduction cascades. Consequently, adducin may be implicated in the development of actin assembly sites (Falet, 2002; Ichetovkin, 2002). Furthermore, signaling cascades that elevate intracellular calcium or activate PKC would destabilize the triadic interaction of adducin, actin filament barbed end, and spectrin, thereby potentially triggering perturbations in the spectrin network (Barkalow, 2003 and references therein).
An essential component of normal development is controlling the transition from cell proliferation to differentiation. One such transition occurs during Drosophila oogenesis. In early oogenesis, germ cells undergo mitotic proliferation and contain a specialized organelle called a fusome, whereas later post-mitotic cells differentiate and lose the fusome as F-actin-rich ring canals form. The hu-li tai shao (hts) gene encodes the only Drosophila Adducin, and is a female-sterile mutant that affects both the fusome and ring canals. One Hts protein, Ovhts, is a polyprotein that is cleaved to produce two products, Ovhts-Fus and Ovhts-RC. Whereas Ovhts-Fus localizes to the fusome in mitotic cells, Ovhts-RC localizes to ring canals throughout later oogenesis. An uncleavable version of Ovhts delays the transition from fusome-containing cells to those that have ring canals. Ovhts is the first polyprotein shown to produce proteins that function in separate structures (Petrella, 2007).
Germline development, like many other developmental programs, involves a preliminary stage of mitotic expansion, followed by differentiation into mature cell types. One hallmark of germline development is that the proliferative phase occurs in clusters of cells interconnected by cytoplasmic bridges. In some systems, such as oogenesis in mammals, cytoplasmic bridges disappear early and cells differentiate separately. However, in most known forms of spermatogenesis and in insect oogenesis, cytoplasmic bridges persist and contribute to differentiation. In insect oogenesis, one cell in each cluster becomes the oocyte and is nourished by the nurse cells through cytoplasmic bridges. In all cases, germline cells must undergo specific changes in gene expression, morphology and function when making the transition from proliferation to differentiation (Petrella, 2007).
Drosophila oogenesis is an attractive model to study the transition from germline cell expansion to differentiation and the formation of intercellular bridges called ring canals (RCs). In the most anterior compartment of the ovary, the germarium, germline stem cells (GSCs) undergo division to form another GSC and a daughter cystoblast. The cystoblast undergoes exactly four mitoses with incomplete cytokinesis forming a 16-cell cyst in which cells are connected by arrested cleavage furrows. Once mitosis is complete the cells differentiate into 15 nurse cells and one oocyte. The proliferating cells and differentiated cells can be easily identified through subcellular structures. During proliferation, an organelle called the fusome is present. After mitosis is complete, the fusome begins to disappear and RCs form at the arrested cleavage furrows (Petrella, 2007).
The fusome is derived from a precursor structure called a spectrosome, which is present in GSCs. A portion of the spectrosome is inherited by the daughter cystoblast, after which it grows and branches during cystoblast divisions to form the fusome. Spectrosome- and/or fusome-like structures have been described in the germline of insects, Xenopus and mouse. A potentially analogous structure is in mammalian lymphocytes. The spectrosome and fusome in Drosophila are areas of highly condensed vesicles including modified endoplasmic reticulum. Fusome membranes are associated with a cytoskeleton that includes the Adductin homolog Hu-li tai shao (Hts), α-Spectrin, ß-Spectrin, Filamin (Cheerio - Flybase), a Spectraplakin homolog named Short Stop (Shot), and Ankyrin. Hts and α-Spectrin are necessary for fusome structure because in mutants the fusome is absent causing dramatic defects in nurse cell number and oocyte specification (Petrella, 2007).
RCs allow the movement of essential proteins and RNA from nurse cells into the oocyte; thus, mutations that result in their disruption are female sterile. Formation of RCs at arrested cleavage furrows follows an organized pattern of RC protein accumulation. F-actin, Filamin and Ovhts-RC, a novel product of the hts locus, begin to accumulate on RCs starting immediately after mitosis ends. Mutations in the gene for Filamin (cheerio) or hts result in a failure of F-actin accumulation and arrested RC development (Petrella, 2007).
Antibodies made against Hts proteins label the fusome and RCs in the germline as well as the follicle cell membranes (Robinson, 1994; Zaccai, 1996a). Mutant phenotypes are consistent with expression data and include loss of oocyte specification, too few nurse cells, and RC deformities (Yue, 1992). Protein localization and mutant phenotype analyses were done prior to knowing that the hts gene produces multiple proteins; therefore, further characterization of hts is needed to understand its roles during oogenesis (Petrella, 2007).
Detailed characterization of the localization of Hts proteins was carried out, focusing on the germline-specific proteins Ovhts and ShAdd. Remarkably, Ovhts is a polyprotein that is cleaved to form two functional products called Ovhts-Fus and Ovhts-RC. Each Ovhts protein is stable during different developmental stages, with Ovhts-Fus present in proliferating cells and Ovhts-RC in post-mitotic differentiating cells. Although other polyproteins have been described, this is the first example where the products are maintained in different developmental time points and subcellular locations. Further experiments with both wild-type and uncleavable Ovhts proteins demonstrate that there is a developmental link between the degradation of the fusome and RC establishment. Finally, Ovhts-RC is shown to be functionally required in RCs throughout oogenesis (Petrella, 2007).
The Drosophila hts gene is known to play key roles in early oogenesis (Lin, 1994; Yue, 1992); yet its exact functions have not been characterized completely. This study has provided evidence that Ovhts is made as a full-length precursor protein that is cleaved, allowing the N-terminal Adducin-like domain (Ovhts-Fus) to localize to the fusome and the C-terminal unique domain (Ovhts-RC) to localize to RCs. Ovhts-Fus is stable only in proliferating cells whereas Ovhts-RC is present only after mitosis is complete, which further emphasizes the importance of post-translational regulation for this protein. An uncleavable form of the protein was created; it can dominantly delay early egg chamber development. Finally, expression of transgenes at specific developmental time points shows that Ovhts-RC is necessary for F-actin localization to RCs during all stages of oogenesis (Petrella, 2007).
The data demonstrate that Ovhts is made as a full-length precursor protein, which is cleaved to form two mature proteins with separate functions. The detection of cleavage products by western blotting and the differential localization of the cleaved proteins in egg chambers provide strong support for this conclusion. The alternative mechanism of producing the downstream protein, Ovhts-RC, by the use of an internal ribosomal entry site (IRES) was ruled out. There is no evidence of an IRES in the sequence of the ovhts mRNA. Furthermore, both of the new hts alleles described in this paper have point mutations 5' to any potential IRES for Ovhts-RC translation, yet neither mutant produces Ovhts-RC (Petrella, 2007).
Endoproteolytic cleavage is a common method of protein regulation. A number of proteins are made as inactive pro-proteins that are activated by cleavage. In these cases part of the protein is removed and degraded to allow the now mature protein to function. In contrast, both mature Ovhts proteins are functional after cleavage, making Ovhts more analogous to polyproteins, which are translated as a single polypeptide and subsequently cleaved into different proteins. Two types of polyproteins have been extensively described: those of some RNA viruses and prohormones such as vasopressin. Viral polyproteins are translation products of whole viral genomes, including both replication enzymes and structural proteins. Viral polyproteins undergo a number of different cleavages, some of which use polyprotein-encoded proteases, and some of which occur through autoproteolysis. The Vasopressin polyprotein includes vasopressin, neurophysin (NP) and a third protein of unknown function. The products of the Vasopressin polyprotein are released by at least two different uncharacterized cellular endoproteases (Petrella, 2007).
The Ovhts polyprotein is likely to be cleaved by an endoprotease rather than undergoing autoproteolysis. The active site for autoproteolysis contains one of three amino acids: Ser, Thr and Cys. The Ovhts cleavage region contains one Ser residue, which when mutated to either a Cys or Ala did not affect Ovhts cleavage. This suggests Ovhts is not cleaved autoproteolytically. Therefore, the more likely mechanism of Ovhts cleavage is through a cellular endoprotease. Because cleavage can occur in S2 cells as well as the ovary, the protease is likely to be widely expressed; however, the cleavage region does not contain any known protease cleavage sites (Petrella, 2007).
The arrangement of the Ovhts polyprotein is conserved in Drosophila. Twelve Drosophila species have been sequenced to date. The hts homolog from each species was annotated, and it was found that the overall gene organization was highly conserved. In all cases, an exon encoding the novel RC domain is similarly positioned near the 3' end of the gene. Whereas the Adducin domains are greater than 90% identical, the RC domain is only 43% conserved, indicating that it is diverging much more quickly. The RC domain is not present as an independent gene or as part of the Adducin gene of other sequenced insect genomes. Thus, it appears that the Ovhts polyprotein was acquired after the divergence of Drosophila (Petrella, 2007).
To explain the existence of an Ovhts polyprotein, a model is proposed in which the Ovhts polyprotein functions to provide Ovhts-RC at a critical time and place and in sufficient abundance through its linkage to Ovhts-Fus. At all stages of oogenesis the Ovhts full-length precursor is translated and cleaved. In Region 1 of germaria, the Ovhts-Fus product is maintained and localizes to the fusome. However, the Ovhts-RC product cannot be detected in Region 1, indicating that it is rapidly degraded. Starting in Region 2, the Ovhts-RC domain begins to be maintained and localizes to RCs. This coincides with the completion of mitotic cell cycles, and perhaps also the expression of an Ovhts-RC binding partner required for its stability. Ovhts-RC first appears as puncta around the fusome, suggesting that the cleavage of the polyprotein may take place on or near the fusome. Another possibility is that the Ovhts-RC stabilizing factor is itself associated with the fusome. In either case the Ovhts-RC protein is in close proximity to the arrested cleavage furrows where it is needed to initiate RC development (Petrella, 2007).
An open question is why the transgenes tested do not restore the fusome in hts mutants. The effects of secondary mutations was ruled out by using several combinations of hts alleles from different backgrounds (i.e. P-element insertions, point mutations and deficiencies), and the existence of another protein isoform was deem unlikely since 3' RACE was performed and only the known transcripts were found. The most likely possibility is that expression of ovhts is required at a specific time early in development for the formation and stability of spectrosomes in germline progenitor or stem cells, and this expression pattern was not recapitulated in the rescue experiments (Petrella, 2007).
The presence of the uncleavable Ovhts-Delta3::GFP protein affects the transition from a fusome to an RC, even though it localizes to both structures. Therefore, cleavage is not required for localization but is necessary for the full function of the Ovhts polyprotein products. The expression of uncleavable Ovhts-Delta3::GFP causes the aberrant persistence of fusome material in RCs. Strikingly, the fusome material includes endogenous wild-type Ovhts-Fus protein that has failed to degrade. Since it is known that mammalian Adducins function as heterotetramers, a direct interaction of wild-type Ovhts-Fus or ShAdd with Ovhts-Delta3::GFP may occur and result in pulling fusome material into the lumen of the RCs. This hypothesis is supported by the fact that RCs in hts mutants that lack wild-type Ovhts-Fus are rescued by Ovhts-Delta3::GFP and do not have fusome plugs or misshapen rims. The dominant-negative effect of Ovhts-Delta3::GFP on wild-type RCs disappears by stage 3, suggesting that uncleaved Ovhts is tolerated in egg chambers that have degraded the fusome. Therefore, it is the transition from fusomes to RCs that is particularly sensitive to the presence of uncleaved Ovhts (Petrella, 2007).
RC formation has been described as a multi-step process beginning with the arrested cleavage furrow followed by the recruitment of Filamin, Ovhts-RC and F-actin, and completed with the addition of other actin binding proteins. It was found that the recruitment of Ovhts-GFP to RCs is delayed in hts mutants lacking a fusome, supporting a role for the fusome (and possibly prelocalization of Ovhts-RC) in the initiation of RC development. Furthermore, without continual expression of Ovhts-RC, F-actin is lost from the inner rim of RCs. Since the F-actin cytoskeleton of RCs is highly dynamic, the absence of Ovhts-RC in hts mutant RCs may tip the balance of actin dynamics toward depolymerization, resulting in the depletion of RC F-actin. Thus, Ovhts-RC is needed continuously either to stabilize polymerized actin or to promote actin polymerization (Petrella, 2007).
hts was isolated as a female sterile mutant (Ding, 1993; Yue, 1992), and products of the hts gene are present on both the fusome and RCs, making Hts an attractive candidate for participating in the transition from fusome-containing proliferating cells to RC-containing differentiating cells. Subsequent work showed that there are four distinct proteins made from splice variants of the hts gene in the ovary (Whittaker, 1999). All predicted Hts proteins share identical N-terminal Head and Neck domains homologous to mammalian Adducin proteins, but have unique C-termini. Add1 and Add2 contain the Adducin Tail and MARCKS domains. ShAdd protein has 23 novel C-terminal amino acids and no MARCKS domain. Ovhts contains 80% of the Adducin Tail domain, and a large C-terminal domain unique to Drosophila Adducins that is called the Ring Canal domain (Petrella, 2007).
Although the predicted size of the Ovhts protein is 128 kDa, ovary extract never contained a protein of this size. Instead, germline specific htsF and 1B1 (detecting the N-terminus) antibodies detected a doublet of ~90 kDa, which are the Add1/2 proteins. When protein extracts were analyzed from virgin female ovaries, which contain fewer late-stage egg chambers and therefore are enriched for germarial tissue, a band of ~80 kDa was detected. As reported previously (Robinson, 1994), the htsRC antibody detects a ~60 kDa doublet. These results suggested that Ovhts is cleaved into two smaller proteins. Moreover, when ovary extracts of flies expressing Ovhts::GFP or Cer::Ovhts were blotted with anti-GFP antibody, only bands that would represent the cleavage products (with the added GFP tag) were seen. These results, along with protein localization data, show that Ovhts is cleaved. Based on the fact that antibodies to the N-terminus of Ovhts label fusomes and antibodies to the C-terminus label RCs, the cleavage products were designated Ovhts-Fus and Ovhts-RC, respectively (Petrella, 2007).
To further investigate Ovhts cleavage, Ovhts was expressed in S2 cells, which do not express endogenous Ovhts. Western analysis with either 1B1 to visualize the N-terminus or htsRC to visualize the C-terminus, revealed 80 kDa and 60 kDa proteins like those detected in ovary extracts. Additionally, an ~140 kDa band that represents full-length Ovhts was apparent in S2 cell extract. Since the full-length protein can only be detected when Ovhts is overexpressed in S2 cells and not in ovary extracts, the cleavage process in ovaries must be very efficient (Petrella, 2007).
date revised: 2 July 2007
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