Detlev Buttgereit

Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS)

During myogenesis in Drosophila embryos, a prominent adhesive structure is formed between precursor cells and fusion‐competent myoblasts (fcms). Here, we show that Duf/Kirre and its interaction partners Rols7 (found in founder myoblasts and growing myotubes) and Sns (found in fcms) are organized in a ring‐structure at the contact points of fcms with precursor cells, while cytoskeletal components like F‐actin and Titin are centered in this ring in both cell types. The cytoplasmic protein Blow colocalizes with the actin plugs in fcms after cell adhesion. Furthermore, the requirement of additional as yet unidentified components was demonstrated by using mammalian C2C12 myoblasts. In this study, we propose that the fusion‐restricted myogenic‐adhesive structure (FuRMAS) is pivotal in linking cell adhesion as well as local F‐actin assembly and dynamics to downstream events that ultimately lead to plasma membrane fusion. Moreover, we suggest that the FuRMAS may restrict the area of membrane breakdown. Developmental Dynamics 236:404–415, 2007.

Drosophila Rolling pebbles colocalises and putatively interacts with alpha-Actinin and the Sls isoform Zormin in the Z-discs of the sarcomere and with Dumbfounded/Kirre, alpha-Actinin and Zormin in the terminal Z-discs

The rolling pebbles gene of Drosophila encodes two proteins, one of which, Rols7, is essential for myoblast fusion. In addition, Rols 7 is expressed during myofibrillogenesis and in the mature muscles. Here it overlaps with alpha-Actinin (α-Actn) and the N-terminus of D-Titin/Kettin/Zormin in the Z-line of the sarcomeres. In the attachment sites of the somatic muscles, Rols7 and the immunoglobulin superfamily protein Dumbfounded/Kin of irreC (Duf/Kirre) colocalise. As Duf/Kirre is detectable only transiently, it may be involved in establishing the first contact of the outgrowing muscle fiber to the epidermal attachment site. We propose that Rols7 and Duf/Kirre link the terminal Z-disc to the cell membrane by direct interaction. This is supported by the fact that in yeast two hybrid assays the tetratricopeptide repeat E (TPR E) of Rols7 shows interaction with the intracellular domain of Duf/Kirre. The colocalisation of Rols7 with α-Actn and with D-Titin/Kettin/Zormin in the Z-dics is reflected in␣interactions with different domains of Rols7 in this assay. In summary, these data show that besides the role in myoblast fusion, Rols7 is a scaffold protein during myofibrillogenesis and in the Z-line of the sarcomere as well as in the terminal Z-disc linking the muscle to the epidermal attachment sites.

During Drosophila embryogenesis the β1 tubulin gene is specifically expressed in the nervous system and the apodemes

We determined the in vivo distribution of the beta 1 tubulin from D. melanogaster using isotype specific antibodies. Maternally expressed beta 1 tubulin is incorporated into mitotic spindles. Later in development a strong expression in the CNS is observed. Furthermore, all chordotonal organs and the apodemes are marked by beta 1 tubulin. Nuclear run-on assays and stage specific in vitro transcription showed a zygotic expression of the beta 1 tubulin gene from the extended germ-band stage onwards. Using the P-element system, we identified several elements; upstream between -2.2 kb and the transcription initiation site, elements for low level expression in the CNS are present. In the intron between +0.44 kb and +2.5 kb enhancer elements are located that drive the expression in the chordotonal organs and the apodemes. Between the start site and +0.44 kb (273 bp) and +2.5 kb and the second exon (315 bp), maternal and CNS enhancers result in full level expression of a lacZ-beta 1 reporter gene. We show, that the beta 1 tubulin gene is very early effector gene starting its expression shortly after the commitment of neuroblast cell fate. This gene offers an excellent model system for the identification of neural and apodeme specific transcription factors.

Expression of the β3 tubulin gene (βTub60D) in the visceral mesoderm of Drosophila is dependent on a complex enhancer that binds Tinman and UBX

Abstract The β3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in the visceral mesoderm and is localized in the intron, revealed a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two binding sites, termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax (Ubx), which are required for high-level expression in pPS6 and PS7. Further analysis of the vm1 enhancer revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all parasegments except pPS6/PS7. Gel-retardation and footprint analysis identified a binding site for the homeodomain protein Tinman, which is essential for the specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vm1 enhancer, named elements 3 (e3), and 6 (e6), results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene. Therefore we conclude that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the β3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors including Tinman and UBX.

In Drosophila melanogaster, the Rolling pebbles isoform 6 (Rols6) is essential for proper Malpighian tubule morphology

During myoblast fusion, cell-cell recognition along with cell migration and adhesion are essential biological processes. The factors involved in these processes include members of the immunoglobulin superfamily like Sticks and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling pebbles (Rols). For rolling pebbles, two differentially expressed transcripts have been defined (rols7 and rols6). However, to date, only a muscle fusion phenotype has been described and assigned to the lack of the mesoderm-specific expressed rols7 transcript. Here, we show that a loss of the second rolling pebbles transcript, rols6, which is expressed from the early bud to later embryonic stages during Malpighian tubule (MpT) development, leads to an abnormal MpT morphology that is not due to defects in cell determination or proliferation but to aberrant morphogenesis. In addition, when Myoblast city or Rac are knocked out, a similar phenotype is observed. Myoblast city and Rac are essentially involved in the development of the somatic muscles and were proposed to be interaction partners of Rols7. Because of the predicted structural similarities of the Rols7 and Rols6 proteins, we argue that genetic interaction of rols6, mbc and rac might lead to proper MpT morphology. We also propose that these interactions result in stable cell connections due to rearrangement of the cytoskeleton.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell-cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles.

Role of the Actin Cytoskeleton Within FuRMAS During Drosophila Myoblast Fusion and First Functionally Conserved Factors in Vertebrates

The larval musculature of Drosophila arises by fusion of two types of myoblasts: the founder cells (FCs), which determine the identity of every individual muscle, and fusion competent myoblasts (FCMs). Cell–cell recognition and adhesion is mediated by the Ig class of transmembrane proteins. They form an adhesion ring/belt at the contact sites of FCMs and FCs/growing myotubes to establish a Fusion Restricted Myogenic Adhesive Structure (FuRMAS). FuRMAS are postulated to trigger myoblast fusion, with the formation, and dissolution of F-actin foci/plugs at the sites of cell–cell contact. Electron-dense vesicles accumulate at opposing membranes of FCMs and FCs/growing muscles, and form a pre-fusion complex (1 μm²). This is hypothesised to take place in the centre of the FuRMAS. The vesicles are thought to be exocytosed, followed by membrane vesiculation and removal of membrane remnants to achieve cytoplasmic continuity over an area of 12 μm². The FCM can then be integrated into the growing myotube. This last step depends on Arp2/3 mediated F-actin reorganisation. The data on cell adhesion, signalling and actin regulation in zebrafish, C2C12 cells and mice strongly indicate conserved factors and principles between Drosophila and vertebrate myoblast fusion.

Drosophila Swiprosin-1/EFHD2 accumulates at the prefusion complex stage during Drosophila myoblast fusion.

In the Drosophila embryo, transient cell adhesion during myoblast fusion is known to lead to the formation of fusion-restricted myogenic-adhesive structures (FuRMASs). Here, we report that within these FuRMASs, a Drosophila homologue of human and mouse swiprosins (EF-hand-domain-containing proteins) is expressed, which we named Drosophila Swiprosin-1 (Drosophila Swip-1). Drosophila Swip-1 is highly conserved and is closely related to the calcium-binding proteins swiprosin-1 and swiprosin-2 that have a role in the immune system in humans and mice. Our study shows that Drosophila Swip-1 is also expressed in corresponding cells of the Drosophila immune system. During myoblast fusion, Drosophila Swip-1 accumulates transiently in the foci of fusion-competent myoblasts (FCMs). Both the EF-hand and the coiled-coil domain of Drosophila Swip-1 are required to localise the protein to these foci. The formation of Drosophila Swip-1 foci requires successful cell adhesion between FCMs and founder cells (FCs) or growing myotubes. Moreover, Drosophila Swip-1 foci were found to increase in number in sing22 mutants, which arrest myoblast fusion after prefusion complex formation. By contrast, Drosophila Swip-1 foci are not significantly enriched in blow2 and ketteJ4-48 mutants, which stop myogenesis beyond the prefusion complex stage but before plasma membrane merging. Therefore, we hypothesise that Drosophila Swip-1 participates in the breakdown of the prefusion complex during the progression of myoblast fusion.

Expression of β1 tubulin (βTub56D) in Drosophila testis stem cells is regulated by a short upstream sequence while intron elements guide expression in somatic cells

Stem cell differentiation to mature spermatozoa is a morphogenetic process that is highly dependent on microtubular arrays. In the early, mitotically active stages of spermatogenesis, only the β1 tubulin isotype is expressed. Analysis of transgenic flies containing β1-lacZ gene fusions revealed that this expression is regulated by sequences located between positions −45 and −191 upstream of the transcription initiation site. Furthermore, β1 tubulin is a major component of cyst cells. Expression in these cells is driven by enhancer elements located in the β1 tubulin gene intron. These enhancer elements also guide expression in combination with the hsp70 basal promoter. In addition, redundant enhancer elements in the intron drive expression in the testis wall. Our data show that within a single tissue, the male gonad, expression of the β1 tubulin gene is under cell-type-specific control mediated by independent cis-acting elements. Therefore in the germ line, control of β1 tubulin expression is strictly governed by promoter-proximal elements, while for the somatic parts of the testis, enhancer elements confer less stringent expression control.

Related enhancers in the intron of the beta1 tubulin gene of Drosophila melanogaster are essential for maternal and CNS-specific expression during embryogenesis.

Expression of the beta1 tubulin gene of Drosophila melanogaster is under complex developmental control. For high levels of transcription in the embryonic central nervous system (CNS) different modules dispersed over 3 kb have to co-operate. Combination of a core promoter with either far upstream localized enhancer elements or, alternatively, with an enhancer from the intron results in expression limited to only a few neuronal cells. Cooperation of all three modules, however, leads to high level expression in most neuronal cells of the CNS. In the intron, we identified a 6 bp core element which is essential for transcription in the CNS, as well as an 8 bp element required for maternal expression. Interestingly, both motifs are quite similar, with CAAAAT as the CNS core and CAAAAAT as the maternal enhancer core. Specific binding of proteins from nuclear extracts to the CNS-specific element could be demonstrated. We suggest that the beta1 tubulin gene represents an ideal marker gene to elucidate connections between pro-neural or neurogenic genes and downstream target genes throughout the CNS.