Cell Biology: Spectrin Tetramer Formation is not Required for Viable Development in Drosophila Mansi R Khanna, Floyd J Mattie, Kristen C Browder, Megan D Radyk, Stephanie E Crilly, Katelyn J Bakerink, Sandra L Harper, David W Speicher and Graham H Thomas J. Biol. Chem. published online November 7, 2014 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Supplemental material: http://www.jbc.org/content/suppl/2014/11/07/M114.615427.DC1.html This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/early/2014/11/07/jbc.M114.615427.full.html#ref-list-1 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 Access the most updated version of this article at doi: 10.1074/jbc.M114.615427 JBC Papers in Press. Published on November 7, 2014 as Manuscript M114.615427 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.615427 Drosophila development without spectrin tetramerization Spectrin tetramer formation is not required for viable development in Drosophila* Mansi R. Khanna1,3, Floyd J. Mattie1, Kristen C. Browder1, Megan D. Radyk1, Stephanie E. Crilly1, Katelyn J. Bakerink1, Sandra L. Harper2, David W. Speicher2 and Graham H. Thomas1,4 1 The Department of Biology and The Department of Biochemistry and Molecular Biology The Pennsylvania State University, University Park, PA, 16802 2 3 Systems Biology Division, The Wistar Institute, Philadelphia, PA, 19104 current address: Center For Neurodegenerative Disease Research, University of Pennsylvania, Philadelphia PA 19104 *Running title: Drosophila development without spectrin tetramerization Keywords: Drosophila, cytoskeleton, cell biology, protein assembly, membrane proteins, spectrin, membrane skeleton α -specR22S, a spectrins. However, tetramerization site mutant homologous to the pathological α -specR28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to β H-spectrin ~1000 fold. Even though spectrins are essential proteins, α specR22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based nonerythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm. Background: Underneath the membrane of most animal cells is a network (membrane skeleton) assembled using tetramers of the protein spectrin. Results: While spectrins are essential proteins, tetramer formation is surprisingly unimportant for Drosophila development. Conclusion: The major roles of the membrane skeleton do not require a conventional network. Significance: The ubiquitous model of the spectrin based membrane skeleton has limited applicability. ABSTRACT The dominant paradigm for spectrin function is that (αβ)2-spectrin tetramers or higher order oligomers form membrane associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type αspectrin binds to both β- and β H-chains with high affinity, resembling other non-erythroid α− and β-Spectrins form (αβ)2 heterotetramers to crosslink F-actin and form spectrin based membrane skeletons (SBMS)5. Analysis of the erythrocyte has provided the dominant paradigm for the SBMS, where short F-actin rods are interconnected by spectrin to form a branching 1 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Downloaded from http://www.jbc.org/ by guest on November 12, 2014 To whom correspondence should be addressed: Graham H Thomas, Department of Biology, Department of Biochemistry and Molecular Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA 16802, USA, Tel.: (814) 863-0716; email; Fax: (814) 865-9131; E-mail: [email protected] Drosophila development without spectrin tetramerization EXPERIMENTAL PROCEDURES Constructs and Spectrin Fragment Purification - Spectrin gene fragments encoding the tetramerization domain were amplified (Table 1) from preexisting cDNA clones (α-spectrin, βspectrin, βH-spectrin) or mutated constructs (αspecR22S), cloned into pGEX4T-1, sequence verified and expressed in E. coli BL21-CodonPlus (DE3)-RIPL (Agilent Technologies, Santa Clara, CA) using standard methods. Bacteria were grown to log phase in LB and induced using 1mM IPTG. Fusion proteins were extracted in Lysis Buffer (50mM Tris.Cl pH8.0, 50mM NaCl, 1mM βmercaptoethanol [βME], 5mM EDTA, 150µM PMSF, 1µg/ml Leupeptin, 1 µg /ml Pepstatin, 1 µg/ml DFP) by sonication on ice. After centrifugation the supernatant was mixed with glutathione-agarose beads (Sigma-Aldrich, St. Louis, MO) that had been previously equilibrated in PBEP (10 mM NaPO4, 130 mM NaCl pH7.3, 1mM βME, 5mM EDTA and 150µM PMSF). After 1hr of tumbling, the beads were packed into a column and washed with ≥10 bed volumes of PBEP, followed by ≥10 bed volumes of PBEP without PMSF, and eluted using G-buffer (50mM Tris.Cl pH8.0, 10mM reduced Glutathione, 1mM 2 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 proteins (20-23), but data on the general requirement for oligmerization via the H2H interaction per se, and therefore conventional network formation dependent upon F-actin crosslinking, is lacking. The analysis of the temperature sensitive (Ts) α-specR22S mutation appeared to indicate that network formation is essential for viability in Drosophila (24). We were interested in utilizing the reported Ts network formation and quantitatively characterized the biochemical consequences of this mutation. We find α-specR22S dramatically affects H2H binding at all temperatures, but that full-length α-spectrinR22S rescues adult development in a null background. Reexamination of the phenotypic consequences of this mutation reveals only minor defects in the ovaries, midgut and at neuromuscular junctions, and that the previously attributed phenotype is actually due to poor fly husbandry. We conclude that tetramerization of α/β-spectrin has very limited roles in fly development, in direct contrast to the red cell paradigm. network resembling a fishing net (1,2). αI/βIspectrin will also form higher order oligomers (heterotetramers, hexamer and octamers etc.) (3) and recent cryoelectron microscopy images have indicated that these are common in the red cell (4). Spectrin oligomerization via the head-to-head (H2H) interaction between the N-terminus of αIspectrin and a C-terminal domain of βI-spectrin is required to form this network. In the erythrocyte, the H2H interaction is of moderate affinity (Kd=400-800nm) (5,6) permitting network formation, but also rearrangement upon shear stress during circulation (7). Mutations that decrease H2H affinity reduce or eliminate oligomerization and cause network disruption and hereditary elliptocytosis or hereditary pyropoikilocytosis (e.g. 6,8,9), so it is clear that the demands for such a network are acute in red cells. H2H binding between vertebrate nonerythroid αΙΙ- and βΙΙ-spectrins is much tighter (Kd=4.5-8.5nm) (5), but the morphology of the non-erythroid SBMS is less well defined. F-actin crosslinking by spectrin has been observed in vitro (10), in the terminal web (11), on the membrane of inner hair cells (12) and in some axons (13). In these cases the models proposed are all based on oligomeric states that form via the H2H interaction, an interaction that has apparently persisted for some 500 Myr (see 14). In most cell types the exact form of the SBMS remains undefined (e.g. 15,16), and other network organizations may exist: Alternative oligomerization mechanisms based on lateral interactions between α-spectrin and β-spectrin have been proposed (17) but remain equivocal (18). Recently, it was shown that human βHspectrin can self associate (19), raising the possibility that F-actin crosslinking could be mediated by this β isoform in the absence of αspectrin. Clearly there is potential for diversity although none has yet been demonstrated. For the purposes of this paper we refer to any H2Hdependent oligomerization process that leads to Factin crosslinking as the ‘conventional network’ – this being the activity upon which all currently visualized networks appear to depend. Genetic investigations have demonstrated that non-erythroid α- and β-spectrins are essential Drosophila development without spectrin tetramerization 3 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 calorimeters (GE Healthcare LifeSciences, Piscataway, NJ). Samples were dialyzed overnight in 10 mM Na PO4, 130 mM NaCl, 1 mM βmercaptoethanol, pH 7.4 before analysis, and wild-type β-spectrin 16-17 at or wild-type βHspectrin 31-32 at 10-15 mM or dialysate buffer (controls) were loaded into the reaction cell. Wildtype or mutant α-spectrin 0-1 at a concentration of 95-185 mM were used as the titrant for experimental measurements and controls at 18°C, 23°C or 29°C stirring at 300 rpm in the reaction cell. Typically, 26 injections of titrant (10 µL each with VP-ITC; 1.5-2µl each with Auto iTC200). Protein concentrations were determined from absorbance at 280nm. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was used to verify the absence of degradation products before and after each experiment. Data were analyzed using OriginLab (v5.0) software as previously described (25,26) to calculate dissociation constants. All interactions were characterized at least three times. Fly strains - The transformation host strain y w was used as a wild-type control. The wild-type UAS-myc-α-spectrin37 strain (27) and lines containing α-speclm32 α-speclm51 and α-speclm111 alleles were a gift from Dr. Ronald Dubreuil (University of Illinois, Chicago, IL). Lines containing α-specrg41 and Df(3L)RR2 were a gift from Dr. Daniel Branton (Harvard University, Cambridge, MA). All of the α-spec alleles were originally generated on a roughoid scarlet ebony chromosome (28); as a result lm/lm or lm/rg allelic combinations exhibit these phenotypes. α−specrg41 is a null allele that is known to be a deletion that results in a frameshift after just 91 residues (20,29,30). α-speclm32 and α-speclm111 have not been sequenced but do not produce detectable protein and are either null or strong hypomorphs (20,29,30). α-speclm51 is lethal over Df(3L)RR2and other α-spec alleles, but is otherwise uncharacterized (20,29,30). The germline MTD-Gal4 driver was obtained from the Bloomington stock center (BL #31777; Indiana University, Bloomington, IN). The αspectrin GFP line contains GFP inserted between amino acids 1330/1 (repeat 12) at the endogenous locus after a weeP GFP-exon-trap insertion (31). βME, 5mM EDTA, 1 µg/ml Pepstatin). Thrombin (Sigma-Aldrich, St. Louis, MO) was dissolved in dH2O, added at an empirically determined ratio to release GST from each spectrin fragment and stopped by the addition of 300µM PMSF. Following extensive dialysis in PBEP the GST/spectrin mixture was again applied to glutathione beads as described above to remove the GST. Flow through from this column was concentrated and residual contaminants were removed by passing it through a preparative HiLoad 16/60 Superdex 75 column. MALDI analysis of each protein verified that the mass of all fragments was as expected (Table 1). Circular dichroism and analytical ultracentrifugation were performed as previously described (6) to verify that each protein had a prototypical helical content for such spectrin fragments and that they behaved as monomers. Proteins were stored on ice until used. HPLC analysis of complex formation – Complex formation between α-spectrin and βspectrin fragments was verified by gel filtration. ~1nmol of each fragment alone or in combination was combined, allowed to assemble on ice (assembly was complete in <1min), then loaded directly on to two analytical (7.8 x 300 mm) TSK gel columns (G3000SWXL and G2000SWXL) in series (Tosoh, King of Prussia, PA) equilibrated in 10 mM Na PO4, 130 mM NaCl, 1 mM EDTA, 0.15 mM PMSF, 1 mM β-mercaptoethanol pH 7.3. Differential scanning calorimetry - Thermal denaturation analyses of spectrin recombinant peptides were performed using an MCS differential scanning calorimeter (MicroCal, Northampton, MA). Purified proteins were exhaustively dialyzed against 10 mM Na PO4, 130 mM NaCl, 1 mM β-mercaptoethanol, pH 7.4. Samples were degassed for 5 minutes, and protein samples at 0.6 to 1.0 mg/mL in the sample cell were compared with dialysate buffer in the reference cell by scanning from 25°C to 100°C at a scan rate of 90°C/h. Data analysis was performed using the ORIGIN (v3.2) software provided with the instrument. Isothermal titration calorimetry Thermodynamic parameters for the binding of the α-spectrin 0-1 recombinant fragments to wild-type 16-17 were determined using a MicroCal VP-ITC or MicroCal Auto-iTC200 isothermal titration Drosophila development without spectrin tetramerization 4 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 supernatant 3A9 (1:1,000-10,000 / 1:20; from The Developmental Studies Hybridoma Bank); mouse anti-c-myc 9E10 (1:100 / 1:10; Enzo Life Sciences, Farmingdale, NY); Alexafluor 488conjugated goat anti-Horseradish Peroxidase (1:500; Jackson ImmunoResearch); Rabbit antiSynaptotagmin Dsyt-CL1 (1:5000; Dr. Noreen Reist, Colorado State University, Fort Collins, CO), mouse anti-Hts (Adducin, 1B1; 1:25) developed by Dr. Howard Lipshitz and mouse anti-Discs large (4F3; 1:50) developed by Dr. Corey Goodman were obtained from The Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. HRP-conjugated anti-mouse secondary antibodies for immunoblots (Jackson Immunoresearch, West Grove, PA) were used at 1:2500. Alexafluor-405, 488 and -594 labeled goat secondary antibodies (Invitrogen, Carlsbad, CA) were preadsorbed against fixed embryos and used at 1:250. Immunoblots used enhanced chemiluminescent substrate (Pierce/Thermo Fisher, Rockford, Il) and were quantified either using a blot scanner (Licor, Lincoln, NE) or with appropriate film exposures (that utilized serial dilutions to verify linear response of the emulsion), followed by imaging and densitometry in Image J (imagej.nih.gov). Immunostaining of ovaries was performed as previously described (33). To visualize F-actin staining in the brush border, guts from wandering third-instar larvae midguts were dissected and accumulated on ice in PBS (130mM NaCl, 10mM Na PO4, pH7.4), then fixed for 20 minutes in 8% formaldehyde in PBS at room temperature. Phalloidin staining was performed as previously described (34). For immunocytochemistry of neuromuscular junctions (NMJ), wandering third instar larvae were dissected leaving the CNS in place and pinned out to expose the body wall muscles, immediately fixed in 4% formaldehyde in PBS for 20 minutes, rinsed with PBS, and the CNS removed and discarded. The remaining body wall muscle “pelt” preparation was unpinned and further processed for immunocytochemistry in glass depression dishes. PBS containing 0.1% Triton X-100 and 5% normal goat serum was used for subsequent antibody incubations and washes. This strain is homozygous viable, exhibits wildtype α-spectrin distributions and was a gift from Dr. Melissa Rolls (Penn State, University Park, PA). All available α-specR22S fly lines no longer expressed α-spectrin (see text). Since, we sought to achieve the same Ts rescue of α-spectrin null mutations shown by Deng et al. we exactly replicated their construct (32). Plasmid P[w+UPS] containing a full-length wild-type αspectrin cDNA (Lee et al., 1993; a gift from Dr. Ronald Dubrueil, University of Illinois, Chicago, IL) was mutated using the QuikChange∆ II XL mutagenesis kit (Agilent Technologies) to create the R22S substitution. In addition, we similarly transformed an NcoI site at the start codon to KpnI to permit cloning into P[w+UM-2], a P-element vector containing the constitutive ubiquitin promoter and Myc tag as previously described (24,32). The entire gene was sequenced after each round of mutagenesis and in the final construct to ensure there were no non-synonymous substitutions. Rainbow Transgenic Flies, Inc. (Camarillo, CA) created fly strains according to standard methods using a y w host. As soon as possible all inserts were combined with a copy of the α-specrg41 null allele to make stocks that should be less susceptible to the effects of chronic α-spectrin overexpression. To create α-specR22S rescued flies the crossing scheme outlined in Figure 3 was followed. With these two crosses rescued flies containing 1, 2 or 3 copies of the α-specR22S transgene were generated and designated 1, 2 or 3 copy rescue (1cr, 2cr, 3cr) respectively. In addition, siblings that contained 1, 2 or 3 copies of the α-specR22S transgene in the presence of one wild-type allele of α-spectrin were generated and designated 1, 2 and three copy coexpression (1cx, 2cx, 3cx) respectively. Rescue to adulthood was quantified as a percentage of the one copy coexpression class in crosses producing an average of 490 offspring (range: 277-721) and included only eclosed and active flies. Antibodies and imaging - The following primary antibodies were used at the dilutions indicated in parentheses (immunoblot / immunofluorescence): one of two preparations of mouse anti-α-Spectrin were used: Ascites #N3 (1:100,000 / 1:10,000; from Dr. Daniel Branton, Harvard University, Cambridge, MA) or culture Drosophila development without spectrin tetramerization RESULTS Characterizing the fly spectrin head-to-head interactions - The α/β-spectrin H2H interaction can be recapitulated using small two-repeat fragments (Fig. 1A) of each subunit (35,36). We cloned and expressed these fragments of the fly α-, β- and βH-spectrin using homologous endpoints (Table 1) (35). The α-specR22S construct that was reported to have a Ts phenotype contained a Myc tag at its N-terminus, but it was not shown whether the presence of the tag was important for this. This addition probably has a minimal effect on the H2H interaction because it resides at the end of an unstructured region Nterminal to the tetramerization site in α-spectrin (35) and is fully functional in rescue experiments (20,29,30). Nonetheless we felt it was important to reproduce the previous construct exactly and therefore included the tag. Solubility was good, purity was high (Fig. 1B) and DSC experiments (Fig. 1C) yielded melting profiles very comparable to published spectrin repeat data (6,37) indicating that they are in native conformation. HPLC analysis indicates that α0-1 forms a complex with both β16-17 and βH31-32 (Fig. 2A). The Kd for these interactions was determined by calorimetry (Fig. 2) (6) and indicates that the wild-type interactions have a Kd (23oC) of 14nM for α0-1/β16-17 and of 5 nM for α0-1/βH31-32 (Fig. 2C). These values are most similar to other non-erythroid α/β interactions (5,6) rather than that of the red cell where the Kd is 400-840 nM (5,6). These results reinforce the notion that the high Kd for red cell α1/βΙ-spectrin binding is a special case. In contrast, α0-1R22S binding to β16-17 is not detectable (Fig. 2B) and binding to βH31-32 is reduced ~1000 fold to 3.7-15µM (Fig. 2B,C), across the reported permissive to non-permissive 5 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 temperature range for the α-specR22S phenotypes (Fig. 2D) (24). While no H2H interaction between α/β-spectrins seems likely in vivo, the residual interaction between α0-1R22S and βH31-32 might permit some tetramer formation depending upon the local spectrin concentration. However, in the red cell, which probably represents an extreme high for an in vivo spectrin concentration, the R28S substitution similarly reduces the Kd (6) and has pathological consequences (38). So it seems unlikely that the residual binding between α0-1R22S and βH31-32 would sustain a wild-type network in vivo. We conclude that the reported Ts phenotype of the α-specR22S allele is not due to a reversible H2H interaction. α-spectrinR22S rescues α-spectrin null flies to adulthood- Existing stocks expressing α-specR22S were made in the mid 1990’s (24), no longer express Myc-tagged α-spectrin and contain promoter-deleted transgenes, suggesting that longterm constitutive expression of this protein has been selected against in the stocks (not shown). We therefore regenerated exactly the same construct starting with the same full-length α-spectrin cDNA (24) including the Myc tag, which might participate in the reported temperature sensitivity. To try to protect the construct from eventual deletion, we immediately crossed each new line into a background heterozygous for the α-specrg41 null allele. The crossing schemes shown in Figure 3 were used to generate rescued flies. In this scheme flies were produced that express up to 3 copies of the transgene in an α-spectrin mutant background (referred to as 1-3 copy rescue flies). Sibling flies heterozygous for each mutant allele containing the rescue construct are referred to as 1-3 copy coexpression flies. Rescued flies were readily obtained at 18, 25 and 29°C and with 1, 2 and in some cases 3 copies of the transgene using the αα-specrg41/α-speclm51, αspecrg41/α-speclm111, rg41 lm32 rg41 R2 spec /α-spec and α spec /Df(3l)R mutant backgrounds. The third chromosome α-specR22S transgene was sufficient to rescue to about 50% of Mendelian expectation (55.4 ± 14.6%; n=7) while the addition of the second chromosome transgene raised this to almost 100% (98.3% ± 28.7%; n=3), although the frequency was highly variable in both cases. Flies were normal in morphology, and we Samples were imaged on a CARV II spinning disc confocal (BD Biosystems, Rockville, MD) with a Retiga EXi camera (Q Imaging systems, Surrey, BC) mounted on an Olympus BX50 microscope (Olympus America, Center Valley, PA) and used iVision 4.5 software for control and capture (Biovision, Exton PA). Figures were assembled using Adobe CS4 Design Standard (Adobe Systems Inc., San Jose, CA) Drosophila development without spectrin tetramerization our hands and are identical to chambers degenerating in response to the nutritional checkpoint (Fig. 5) (39). The existence of this checkpoint and the importance of using well-fed females to examine ovaries were not realized until after Deng et al. published their paper (39). We conclude that there is no such phenotype in the αspecR22S rescued females. However, there are more subtle abnormalities in oogenesis not previously described. First, we noticed that fusome structure is altered. The fusome is a cytoplamic membrane ‘network’ that contains α/β-spectrin and Adducin (40), forms at sites of incomplete cytokinesis, associates with spindle poles during cystoblast division, and reaches its greatest complexity in germarium regions 2a/b before starting to disappear. Staining for Adducin (1B1) reveals fusome morphology (Fig. 6A) (see 41). In rescued α-specrg41/α-speclm111 flies 1B1 staining becomes fragmentary in region 2 (Fig. 6B-B’’) suggesting that spectrin tetramerization is required for late fusome integrity. We also see a similar fragmentation when one copy of a wild-type αspectrin gene is present (Fig. 6C-C’) showing that the α-specR22S can dominantly disrupt the fusome. This is not a fixation artifact because fragmentation was also seen with coexpression in an α-spectrin GFP gene trap line when we imaged freshly dissected germaria (Fig. 6D,D’). It is also possible that expression of α-spectrin above wildtype levels is responsible for this phenotype. We therefore overexpressed wild-type α-spectrin in the germline in a wild-type α-spectrin background using the Gal4/UAS system (42) with the MTDGal4 driver. Staining MTD-Gal4>UAS-αspectrin37 ovarioles for 1B1 reveals fusomes of normal complexity (Fig. 6E). The second phenotype seen in the ovaries of rescued females is an occasional gap in the follicle cell monolayer (Fig. 6F-F’’). These are mostly just one or two cell diameters in size, although a handful of much larger ones have been observed. Given the fertility of the rescued females and the low frequency of degenerating chambers we assume that most of these resolve themselves. A number of other tissues are affected by loss of function α-spectrin alleles in the fly. In particular the spectrins are particularly necessary in the nervous system (e.g. 27,43,44) and in the 6 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 do not see evidence of the sluggish behavior in rescued flies reported by Deng et al. (24) (Movie S1). We speculate that the difference between our results and the previous study by Deng et. al. (24) may have arisen because of over- or underexpression from their original transgene insertion(s), which would almost certainly have had different levels of expression from our lines due to position effects. We cannot do a direct comparison since the original transgenic lines were lost; however, we sought to verify that our transgenes were producing α-specR22S at physiological levels by comparison to wild-type lines using quantitative immunoblots of whole fly extracts (Fig. 4). The results appear to indicate that our single and dual copy rescue flies express αspecR22S at ~80% wild-type levels and that coexpression with one copy of wild-type αspectrin results in mild overexpression. However these results are quite variable and do not achieve statistical significance (One sample t-test versus normalized control 0.07<P<0.21, n=7). Since two copies of the transgene produce a better rescue frequency while the size of the stable αspectrinR22S pool does not change, we suggest that the rate of α-spectrin production may influence viability. We conclude that our transgenes are producing near wild-type amounts of α-specR22S protein. Probing with the anti-Myc antibody for the tagged α-specR22S protein reveals that the presence of wild-type α-spectrin often appears to cause the selective destabilization of α-specR22S (Fig. 4C) or that the ability to form tetramers results in the selective stabilization of wild-type α-spectrin. However, this result is also highly variable and although striking on some blots, again does not reach statistical significance (One sample t-test versus normalized control 0.09<P<0.15, n=6). α-spectrinR22S rescued α-spectrin null flies exhibit subtle tissue abnormalities- Rescued males and females raised at any temperature mated and viable eggs are produced, indicating that early development (prior to zygotic α-spectrin transcription) also does not require tetramerization. We examined the ovaries of rescued flies closely because of the previously reported degeneration phenotype in post-stage 7 egg chambers (24). Such chambers were rare in Drosophila development without spectrin tetramerization length proteins in vivo. Of course, it is formally possible that in vivo conditions (e.g. local concentration or interaction with other proteins) could lead to tetramerization despite the presence of the R22S substitution. However, the magnitude of the effects of this mutation on tetramerization are similar to that seen in the homologous mutation in red cell spectrin, where the effective spectrin concentration has been estimated to be as high as 230mg/ml (~380µM) (47), and yet an erythrocyte pathology still results. Therefore, the simplest explanation for our results is that the functionality of a conventional red cell-type network is not that important in fly development in any vital tissue. Previous results suggesting that a conventional network involving spectrin crosslinking of F-actin might not be so important in non-erythroid tissues came from examination of dominant H2Hdisrupting mutations in βΙ-spectrin that lead to hereditary elliptocytosis and hereditary pyropoikillocytosis (see 48 and references therin). Despite the expression of the non-erythroid βIΣII spliceoform of this protein in other tissues, these dominant effects are confined to the red cell. Homozygosity for the βΙ-spectrinProvidence allele in the tetramerization domain is lethal (49), but other tissues, notably muscles, nonetheless develop normally in affected individuals (50). However, the issue of isoform redundancy could not be addressed in this study since βIII- and βIVspectrins were unknown at the time. In the fly, several papers suggest that βspectrin function in neurons and epithelial cells is independent of α-spectrin and need not therefore depend upon a conventional network (21,51,52). Also in fly, the W2033R mutation in the βspectrin tetramerization domain was reported to rescue β-spec mutant flies (53) although this was not backed up with a biochemical demonstration that the mutation had the homologous effect. Vertebrate β-spectrin unaccompanied by αspectrin can be found in high molecular weight complexes (54), but it is not clear whether these are oligomers or complexes with other proteins. In all these cases there is currently no published data that indicates whether β-spectrin that is not bound to α-spectrin is in any kind of network or is operating in monomeric or oligomeric form. DISCUSSION The wild-type dissociation constants for the tetramerization reaction between fly α/β-spectrin and α/βH-spectrin are both in the low nM range and about 100 fold lower than that of red cell spectrin. This is in line with all other results for non-erythroid spectrins to date (5), and is consistent with the notion that the lower tetramerization affinity associated with red cell spectrin results from the specialized nature of this cell type. Our biochemical data strongly suggests that tetramerization will not occur between αspectrinR22S and β-spectrin in vivo, and that the interaction is severely αR22S/βH-spectrin compromised. The analysis of the recombinant peptides used to determine the binding affinities between the α-spectrin and β/βH spectrin tetramerization domains represents an accepted model since its inception over 20 years ago (45), and analyses of larger proteolytic fragments or full-length proteins has yielded the same properties and binding affinities as the short fragments used here (46). Furthermore, our results are fully in line with other estimates of nonerythroid spectrin tetramerization interactions (5). This strongly suggests that our measurements reflect the tetramerization potential of the full- 7 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 midgut (34). We therefore performed initial phenotypic analyses on the morphology of the neuromuscular junction (NMJ) and brush border of rescued third instar larvae. In NMJ of rescued 3rd instar larvae all our marker proteins (including α-spectrin) localized normally (Fig. 6H-J). From the location and complexity of the NMJ they reveal, we further infer that the motor neurons find and invade the musculature normally with little difficulty. However, we do see that the NMJ itself has a slightly altered morphology, with some of the boutons in rescued larvae often being less individuated (Fig. 6I,J). Similarly, phalloidin staining of the brush border of the middle midgut reveals no conspicuous abnormalities (Fig. 6 K,L). The role of tetramerization at both of these locations, is therefore not of sufficient importance to compromise the tissue under culture conditions. In future studies it will be interesting to see if any specific functionalities at either of these locations is perturbed by the absence of tetramerization. Drosophila development without spectrin tetramerization 8 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 However, the selective advantage need only be small over this period of time to result in the present day high affinity interaction. It will be of considerable interest for future studies to identify what changes in functionality are present (e.g. at the NMJ) in the rescued fly background that underlie this selective pressure. Our results change our perspective on these essential proteins. The red cell model and its variants in the terminal web and axons all depend upon F-actin crosslinking by spectrin oligomers that are mediated by the H2H interaction and are widely assumed to apply in all non-erythroid tissues. While there is a role for a network of this type, our results clearly indicate that this is not perhaps the core function of spectrin in nonerythroid tissue, and further reinforces the notion that the dependence of the red cell on a fully networked SBMS is a special case. Several studies indicate a significant role for spectrin in endomembrane trafficking (55-59). In this context our results make sense, given that the size of most transport organelles is far too small to accommodate a traditional spectrin network. It has been suggested that αΙ/βΙ-spectrin might form oligomeric structures without the H2H interaction (3) and it was recently shown that human βH-spectrin can self associate (19). Such results raise the possibility that non-conventional networks may exist, and clearly their role would not be tested by the current work. The relative contribution of conventionally networked spectrin to all the tissues in an organism is directly assayed by this study for the first time because all of the α-spectrin in the fly is affected by the R22S substitution- there are no other isoforms present. Since a viable and functional adult is produced, H2H oligomerization is by definition not a vital function, but since we do detect minor morphological defects in several tissues, it would appear that there is a role for the conventional network, one of refinement. In the fly’s normal environment, the role of H2H oligomerization must be sufficiently significant to provide a selective pressure for the maintenance of this interaction. Since the structure of the spectrins, including the tetramerization domains, has been maintained for some 500 Myr (14), there is clearly selective pressure for its maintenance. Drosophila development without spectrin tetramerization REFERENCES 1. 2. 3. 4. 5. 6. 7. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 9 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 8. Elgasaeter, A., Stokke, B. T., Mikkelsen, A., and D., B. (1986) The molecular basis of erythrocyte shape. Science 234, 1217-1222 Mohandas, N., and Gallagher, P. G. (2008) Red cell membrane: past, present, and future. Blood 112, 3939-3948 Morrow, J. S., and Marchesi, V. T. (1981) Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton. J. Cell Biol. 88, 463-468 Nans, A., Mohandas, N., and Stokes, D. L. (2011) Native ultrastructure of the red cell cytoskeleton by cryo-electron tomography. Biophys. J. 101, 2341-2350 Bignone, P. A., and Baines, A. J. (2003) Spectrin alpha II and beta II isoforms interact with high affinity at the tetramerization site. Biochem. J. 374, 613-624 Gaetani, M., Mootien, S., Harper, S., Gallagher, P. G., and Speicher, D. W. (2008) Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site. Blood 111, 5712-5720 An X, L. M., Chasis JA, Mohandas N, Gratzer W. (2002) Shear-response of the spectrin dimertetramer equilibrium in the red blood cell membrane. J. Biol. Chem. 277, 31796-31800 Garbarz, M., Tse, W. T., Gallagher, P. G., Picat, C., Lecomte, M. C., Galibert, F., Dhermy, D., and Forget, B. G. (1991) Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation. J. Clin. Invest. 88, 76-81 Gallagher, P. G., Petruzzi, M. J., Weed, S. A., Zhang, Z., Marchesi, S. L., Mohandas, N., Morrow, J. S., and Forget, B. G. (1997) Mutation of a highly conserved residue of betaI spectrin associated with fatal and near-fatal neonatal hemolytic anemia. J. Clin. Invest. 99, 267-277 Dubreuil, R. R., Byers, T. J., Stewart, C. T., and Kiehart, D. P. (1990) A beta-spectrin isoform from Drosophila (beta H) is similar in size to vertebrate dystrophin. J. Cell Biol. 111, 1849-1858 Hirokawa, N., Cheney, R. E., and Willard, M. (1983) Location of a protein of the fodrin-spectrinTW260/240 family in the mouse intestinal brush border. Cell 32, 953-965 Wada, H., Kimura, K., Gomi, T., Sugawara, M., Katori, Y., Kakehata, S., Ikeda, K., and Kobayashi, T. (2004) Imaging of the cortical cytoskeleton of guinea pig outer hair cells using atomic force microscopy. Hear. Res. 187, 51-62 Xu, K., Zhong, G., and Zhuang, X. (2013) Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452-456 Thomas, G. H., Newbern, E. C., Korte, C. C., Bales, M. A., Muse, S. V., Clark, A. G., and Kiehart, D. P. (1997) Intragenic duplication and divergence in the spectrin superfamily of proteins. Mol. Biol. Evol. 14, 1285-1295 Morone, N., Fujiwara, T., Murase, K., Kasai, R. S., Ike, H., Yuasa, S., Usukura, J., and Kusumi, A. (2006) Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. J. Cell Biol. 174, 851-862 Hartwig, J. H., and Yin, H. L. (1988) The organization and regulation of the macrophage actin skeleton. Cell Motil. Cytoskeleton 10, 117-125 Morrow, J. S., Speicher, D. W., Knowles, W. J., Hsu, C. J., and Marchesi, V. T. (1980) Identification of functional domains of human erythrocyte spectrin. Proc Natl Acad Sci U S A 77, 6592-6596 An, X., Guo, X., Yang, Y., Gratzer, W. B., Baines, A. J., and Mohandas, N. (2011) Inter-subunit interactions in erythroid and non-erythroid spectrins. Biochim. Biophys. Acta 1814, 420-427 Papal, S., Cortese, M., Legendre, K., Sorusch, N., Dragavon, J., Sahly, I., Shorte, S., Wolfrum, U., Petit, C., and El-Amraoui, A. (2013) The giant spectrin betaV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route. Hum. Mol. Genet. 22, 3773-3788 Drosophila development without spectrin tetramerization 20. 21. 22. 23. 24. 25. 26. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 10 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 27. Lee, J. K., Coyne, R. S., Dubreuil, R. R., Goldstein, L. S., and Branton, D. (1993) Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster. J. Cell Biol. 123, 17971809. Dubreuil, R. R., Wang, P., Dahl, S., Lee, J., and Goldstein, L. S. (2000) Drosophila beta spectrin functions independently of alpha spectrin to polarize the Na,K ATPase in epithelial cells. J. Cell Biol. 149, 647-656. Thomas, G. H., Zarnescu, D. C., Juedes, A. E., Bales, M. A., Londergan, A., Korte, C. C., and Kiehart, D. P. (1998) Drosophila betaHeavy-spectrin is essential for development and contributes to specific cell fates in the eye. Development 125, 2125-2134 Austin, J., McKeown, C., and Patel, A. (1995) Sma-1 encodes a spectrin required for morphogenesis of the C. elegans embryo. Mol. Biol. Cell 6, 270a Deng, H., Lee, J. K., Goldstein, L. S., and Branton, D. (1995) Drosophila development requires spectrin network formation. J. Cell Biol. 128, 71-79. Harper, S. L., Begg, G. E., and Speicher, D. W. (2001) Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization. Biochemistry 40, 9935-9943 Li, D., Harper, S., and Speicher, D. W. (2007) Initiation and propagation of spectrin heterodimer assembly involves distinct energetic processes. Biochemistry 46, 10585-10594 Mazock, G. H., Das, A., Base, C., and Dubreuil, R. (2010) Transgene Rescue Identifies an Essential Function for Drosophila {beta} Spectrin in the Nervous System and a Selective Requirement for Ankyrin-2 Binding Activity. Mol. Biol. Cell Sliter, T. J., Henrich, V. C., Tucker, R. L., and Gilbert, L. I. (1989) The genetics of the Dras3Roughened-ecdysoneless chromosomal region (62B3-4 to 62D3-4) in Drosophila melanogaster: analysis of recessive lethal mutations. Genetics 123, 327-336 Lee, J. K., Brandin, E., Branton, D., and Goldstein, L. S. (1997) alpha-Spectrin is required for ovarian follicle monolayer integrity in Drosophila melanogaster. Development 124, 353-362 de Cuevas, M., Lee, J. K., and Spradling, A. C. (1996) alpha-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 122, 3959-3968 Clyne, P. J., Brotman, J. S., Sweeney, S. T., and Davis, G. (2003) Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements. Genetics 165, 1433-1441 Deng, H. (1995) Biochemical and Functional Characterization of Drosophila Spectrin Tetramer Formation. Ph.D., Harvard University Zarnescu, D. C., and Thomas, G. H. (1999) Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila. J. Cell Biol. 146, 1075-1086. Phillips, M. D., and Thomas, G. H. (2006) Brush border spectrin is required for early endosome recycling in Drosophila. J. Cell Sci. 119, 1361-1370 Ipsaro, J. J., Harper, S. L., Messick, T. E., Marmorstein, R., Mondragon, A., and Speicher, D. W. (2010) Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex. Blood 115, 4843-4852 Kotula, L., DeSilva, T. M., Speicher, D. W., and Curtis, P. J. (1993) Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site. J. Biol. Chem. 268, 14788-14793 An, X., Zhang, X., Salomao, M., Guo, X., Yang, Y., Wu, Y., Gratzer, W., Baines, A. J., and Mohandas, N. (2006) Thermal stabilities of brain spectrin and the constituent repeats of subunits. Biochemistry 45, 13670-13676 Coetzer, T. L., Sahr, K., Prchal, J., Blacklock, H., Peterson, L., Koler, R., Doyle, J., Manaster, J., and Palek, J. (1991) Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis. J. Clin. Invest. 88, 743-749 Soller, M., Bownes, M., and Kubli, E. (1999) Control of oocyte maturation in sexually mature Drosophila females. Dev. Biol. 208, 337-351 Drosophila development without spectrin tetramerization 40. 41. 42. 43. 44. 45. 46. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 11 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 47. Lighthouse, D. V., Buszczak, M., and Spradling, A. C. (2008) New components of the Drosophila fusome suggest it plays novel roles in signaling and transport. Dev. Biol. 317, 59-71 de Cuevas, M., and Spradling, A. C. (1998) Morphogenesis of the Drosophila fusome and its implications for oocyte specification. Development 125, 2781-2789 Brand, A. H., and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415 Pielage, J., Fetter, R. D., and Davis, G. W. (2005) Presynaptic spectrin is essential for synapse stabilization. Curr. Biol. 15, 918-928 Pielage, J., Fetter, R. D., and Davis, G. W. (2006) A postsynaptic spectrin scaffold defines active zone size, spacing, and efficacy at the Drosophila neuromuscular junction. J. Cell Biol. 175, 491503 Kotula, L., DeSilva, T. M., Speicher, D. W., and Curtis, P. J. (1993) Functional characterization of recombinant human red cell alpha- spectrin polypeptides containing the tetramer binding site. J. Biol. Chem. 268, 14788-14793. DeSilva, T. M., Peng, K. C., Speicher, K. D., and Speicher, D. W. (1992) Analysis of human red cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides. Biochemistry 31, 10872-10878 Lux, S. E. (1979) Spectrin-actin membrane skeleton of normal and abnormal red blood cells. Semin. Hematol. 16, 21-51 Gallagher, P. G., and Forget, B. G. (1996) Hematologically important mutations: spectrin variants in hereditary elliptocytosis and hereditary pyropoikilocytosis. Blood Cells Mol. Dis. 22, 254-258 Gallagher, P. G., Weed, S. A., Tse, W. T., Benoit, L., Morrow, J. S., Marchesi, S. L., Mohandas, N., and Forget, B. G. (1995) Recurrent fatal hydrops fetalis associated with a nucleotide substitution in the erythrocyte beta-spectrin gene. J. Clin. Invest. 95, 1174-1182 Weed, S. A., Stabach, P. R., Oyer, C. E., Gallagher, P. G., and Morrow, J. S. (1996) The lethal hemolytic mutation in beta I sigma 2 spectrin Providence yields a null phenotype in neonatal skeletal muscle. Lab. Invest. 74, 1117-1129 Garbe, D. S., Das, A., Dubreuil, R. R., and Bashaw, G. J. (2007) beta-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in the Drosophila embryonic CNS. Development 134, 273-284 Hulsmeier, J., Pielage, J., Rickert, C., Technau, G. M., Klambt, C., and Stork, T. (2007) Distinct functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding. Development 134, 713722 Das, A., Base, C., Dhulipala, S., and Dubreuil, R. R. (2006) Spectrin functions upstream of ankyrin in a spectrin cytoskeleton assembly pathway. J. Cell Biol. 175, 325-335 Porter, G. A., Scher, M. G., Resneck, W. G., Porter, N. C., Fowler, V. M., and Bloch, R. J. (1997) Two populations of beta-spectrin in rat skeletal muscle. Cell Motil. Cytoskeleton 37, 7-19 Tjota, M., Lee, S. K., Wu, J., Williams, J. A., Khanna, M. R., and Thomas, G. H. (2011) Annexin B9 binds to beta(H)-spectrin and is required for multivesicular body function in Drosophila. J. Cell Sci. 124, 2914-2926 Lorenzo, D. N., Li, M. G., Mische, S. E., Armbrust, K. R., Ranum, L. P., and Hays, T. S. (2010) Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila. J. Cell Biol. 189, 143-158 Ayalon, G., Hostettler, J. D., Hoffman, J., Kizhatil, K., Davis, J. Q., and Bennett, V. (2011) Ankyrin-B Interactions with Spectrin and Dynactin-4 Are Required for Dystrophin-based Protection of Skeletal Muscle from Exercise Injury. J. Biol. Chem. 286, 7370-7378 Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) Ankyrin-G and beta2-spectrin collaborate in biogenesis of lateral membrane of human bronchial epithelial cells. J. Biol. Chem. 282, 2029-2037 Drosophila development without spectrin tetramerization 59. Kizhatil, K., Davis, J. Q., Davis, L., Hoffman, J., Hogan, B. L., and Bennett, V. (2007) AnkyrinG is a molecular partner of E-cadherin in epithelial cells and early embryos. J. Biol. Chem. 282, 26552-26561 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 12 Drosophila development without spectrin tetramerization ACKNOWLEDGEMENTS The authors would like to thank Peter Hembach for technical assistance, Julia Fecko for assistance with calorimetry at Penn State, Dr. Ronen Marmorstein for emergency use of his calorimeter, Dr. Lorraine Santy for use of her blot imager, members of the Thomas lab for critically reading the manuscript, the Bloomington stock center and investigators who contributed antibodies and fly strains. FIGURE LEGENDS FIGURE 1. Characterization of expressed spectrin fragments. A. Schematic diagram illustrating the location of the tetramerization site in an (αβ)2-spectrin tetramer (pink arrow) above the structure of the vertebrate αI/βI-spectrin interaction at this location. The structure backbone is colored to illustrate the spectrin repeats β16 (yellow), β17 (green), α0 (red) and α1 (blue). The location of the R28 side chain is shown in space filling representation. B. SDS-PAGE analysis of purified proteins and markers. Although the proteins do not migrate in the predicted order, MALDI analysis indicates that all the proteins have the expected molecular weight (Table 1). C. DSC reveals typical spectrin repeat melting profiles (e.g. 6,37) indicating correctly folding. Dashed red lines are fitted curves estimating the melting temperatures at: 59.6, 60.8, 49.0/61.4 and 48.6/75.2oC for α0-1wt, α0-1R22S, βH31-32, β16-17 respectively. DSC experiments were done once, primarily for quality control purposes. FIGURE 2. Complex formation between α- and β-spectrin tetramerization domains. A. HPLC gel filtration profiles. Solid lines show an equimolar mixture of α0-1 and β16-17 (top) or βH31-32 (bottom); dashed lines α0-1 alone; dotted lines β16-17 (top) and βH31-32 (bottom). Complex formation is robust in both cases. B. Representative ITC analyses for binding between α0-1 (αwt) or α0-1α-specR22S (αR22S) and β16-17 (β) or βH31-32 (βH) as indicated. C. Table showing the Kd (with 95% confidence intervals) determined for each interaction at three different temperatures. n – number of replicates; UND – interaction undetectable. The α-specR22S mutation eliminates interaction with β16-17 whereas a reduction of ~1000 fold is seen with βH31-32. FIGURE 3. Genetics of crosses resulting in a-specR22S rescued and coexpressing flies. FIGURE 4. Analysis of α-spectrin levels by quantitative immunoblot. A. An example blot where fly extracts from wild-type, α-spectrinR22S rescued (one or two copies of transgene) and α-spectrinR22S coexpressing (one or two copies of transgene) flies were probed for α-spectrinR22S (via its myc-tag), total α-spectrin and α-tubulin (for normalization). B. Comparison of total α-spectrin levels with wild-type. 13 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 FOOTNOTES *This work was supported by grants MCB-1122013 to GHT from The National Science Foundation and HL038794 to DWS from the National Institutes of Health 1 From The Department of Biology and The Department of Biochemistry and Molecular Biology The Pennsylvania State University, University Park, PA, 16802 2 Systems Biology Division, The Wistar Institute, Philadelphia, PA, 19104 3 The Department of Biochemistry and Molecular Biology Thomas Jefferson University, Philadelphia, PA, 19107 USA 4 To whom correspondence should be addressed: Graham H Thomas, Department of Biology, Department of Biochemistry and Molecular Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA 16802, USA, Tel.: (814) 863-0716; email; Fax: (814) 865-9131; E-mail: [email protected] 5 The abbreviations used are: SBMS – spectrin based membrane skeleton; H2H – head-to-head tetramerization interaction; Ts – temperature sensitive; DSC – differential scanning calorimetry Drosophila development without spectrin tetramerization Rescued flies have ~80% of the α-spectrin seen in wild-type, while coexpressed α-spectrinR22S is not additive with wild-type, probably reflecting the observation that it is destabilized in the presence of wildtype protein (panel C); C. Comparison of α-spectrinR22S levels in coexpressing and rescued flies. There is less α-spectrinR22S when wild-type α-spectrin is present. Genotype rescued: a-specrg41/Df(3L)RR2. Background for coexpression: a-specrg41/TM6B. Error bars represent 95% confidence intervals. FIGURE 5. Degenerating egg chambers in α -specR22S coexpression and rescue females. Staining is for Dlg or α-spectrin as cell shape markers and DAPI to show the characteristic pycnotic nuclei (e.g. arrow in A). A, B. degenerating chambers (asterisks) from one copy coexpression females held at 22°C and 29°C respectively for 4-5 days. C, D. degenerating chambers (asterisks) from one copy rescue females held at 22°C and 29°C respectively for 4-5 days. (Note that the degenerating chamber in C overlies a normal chamber). This phenotype is absolutely typical of degeneration triggered by the stage 8 nutritional checkpoint and occurs at a frequency of less than 10% in all genotypes examined as long as females were well fed on fresh yeast paste for 4-5 days. Scale bars represent 20µm. α 14 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 FIGURE 6. Tissue defects in α -specR22S rescued α-spec mutants. Ovaries: Germaria and egg chambers stained with the 1B1 antibody (A-C’, E-G’) or expressing an α-spectrin GFP protein trap (D, D’). Germarium images are maximum projections of confocal stacks, most of which were digitally tilted to obtain the optimum view. A. Wild-type. Inset shows two fusomes showing the normal branching structure in region 2b. One fusome is indicated by the dashed line. B-B’’. 1, 2 or 3 copy rescue α-speclm111/αspecrg41 flies (1cr, 2cr and 3cr respectively). C-C’. α-speclm111/+(TM6) heterozygous flies coexpressing 2 or 3 copies of α-specR22S (2cx and 3cx respectively). D-D’. αwt – weePα/TM6. α1cx – is weePα/P{αspec -specR22S} α-specRG41. E. Overexpression of wild-type α-spectrin (MTD-Gal4>UAS-α-spectrin37). FF’’. Egg chambers from α-specR22S rescued flies expressing 1, 2 or 3 copies of the α-specR22S transgene stained with 1B1 that exhibit gaps in the normally continuous follicle cell monolayer (arrows, other labels as in B). G-G’’. Germarium and egg chambers from two copy coexpression females stained for the Myc epitope on the α-spectrinR22S protein, which can be seen to be present at the fusome and membrane in an identical distribution to wild-type α-spectrin. Scale bars represent 10µm. Neuromuscular junctions (NMJ): Anti-HRP (HRP) reveals the neuron. Synaptotagmin (SYT) marks the presynaptic side of the boutons; α-spectrin (α-sp) is concentrated in the subsynaptic reticulum on the postsynaptic side of the boutons. H-H’’- wild-type; I-J one copy rescue of α-specRG41/Df(3L)RR2. While rescued NMJ have many regions that appear wild-type (arrows in I’) they often have regions where the boutons are less well individuated (dashed lines in I’, J). As in the ovaries α-spectrinR22S exhibits a wild-type postsynaptic accumulation at the boutons. Scale bars represent 20µm. Midgut: Staining for F-actin reveals the brush borders on the apical domain of the cells. Wild-type cuprophilic cells have a deeply invaginated apical surface with a thin channel out to the central lumen (arrowheads in K) but can also be more open exhibiting a smooth domed surface (insets). Cuprophilc cells have more intense staining from longer microvilli. L,M - in one copy rescue of α-specRG41/Df(3L)RR2 in these larvae there are large variations in cell shape and position, with many malformed and crenellated cuprophilic cell lumens (asterisks); however, a brush border is still present in all cases. Scale bars represent 20µm. Drosophila development without spectrin tetramerization TABLE 1. Spectrin fragments expressed and purified for this study. Spectrin fragment Codons From-To1 Primers (5’-3’)2 Mr Predicted/ MALDI3 α 0-1 2…152 gspefENFT …QQAL gtgaattcGAGAACTTTACACCCAAAGAGGT atgcggccgcttaGAGAGCCTGCTGCAGCTTTAGAC 18441.58/ 18441.41 α myc01R22S Myc…2… R22S…152 gspefEQKL …QQAL gtgaattcGAGCAAAAGCTCATTTCTGAAGA cgctcgagttaGAGAGCCTGCTGCAGCTTTAGAC 19702.0/ 19700.14 β 16-17 1911…2093 gsTGDL …RRQE gtggatccACAGGCGACTTATTCCG cgctcgagttaCTCCTGGCGCCTCTTCATTTCC 21651.61/ 21637.03 gsAEQL gtggatccGCAGAGCAGCTGCAGTCCTACTT …QLEQ cgctcgagttaCTGCTCCAGCTGGTGACGGAATA βH 31-32 3412…3594 21666.50/ 21666.80 15 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 Indicated are the relavant codons included by use of the indicated primers. Myc indicates that a Myc tag was present on this construct. 1Amino acids in lower case were added by the construct and remain present following thrombin cleavage. 2Bases in lower case were added for cloning purposes. The restriction sites used (Eco RI, Not I and Xho I) are underlined in the primers and the added stop codon is in italics. 3 MALDI estimates of the molecular weights of the purified proteins Drosophila development without spectrin tetramerization Figure 1 50 40 30 20 15 10 kcal/mole 10 5 α0-1R22S 5 0 0 30 40 50 60 70 80 90 30 40 50 60 70 80 90 Temperature (oC) Temperature (oC) 20 20 βH31-32 β16-17 15 15 kcal/mole 17 6β1 32 31 βH 22S R -1 wt α0 0-1 α B α0-1wt 10 10 5 5 0 0 30 40 50 60 70 80 90 30 40 50 60 70 80 90 Temperature (oC) Temperature (oC) 16 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 R28 10 kcal/mole C kcal/mole A Drosophila development without spectrin tetramerization Figure 2 25 20 B αwt β αR22S β α+β mV 15 10 5 0 25 20 25 30 Elution time (min) βH 35 α + βH C mV 15 10 5 0 25 30 Elution time (min) 35 17 αwt βH αR22S βH Downloaded from http://www.jbc.org/ by guest on November 12, 2014 A Drosophila development without spectrin tetramerization Figure 3. Parental genotypes Relevant progeny genotypes CyO, Cy + + ; α−speclm111 TM6B, Tb Hu e X P[Ubi-α−specR22S] CyO, Cy ; α−specRG41 P[Ubi-α−specR22S] TM6B, Tb Hu e P[Ubi-α−specR22S] + CyO, Cy + ; α−specRG41 P[Ubi-α−specR22S] TM6B, Tb Hu e P[Ubi-α−specR22S] ; TM6B, Tb Hu e X P[Ubi-α−specR22S] CyO, Cy ; α−speclm111 TM6B, Tb Hu e α−speclm111 α−speclm111 TM6B, Tb Hu e P[Ubi-α−specR22S] CyO, Cy CyO, Cy α−specRG41 P[Ubi-α−specR22S] ; + α−specRG41 P[Ubi-α−specR22S] α−speclm111 P[Ubi-α−specR22S] + P[Ubi-α−specR22S] P[Ubi-α−specR22S] ; α−specRG41 P[Ubi-α−specR22S] P[Ubi-α−specR22S] ; ; ; α−specRG41 P[Ubi-α−specR22S] TM6B, Tb Hu e α−specRG41 P[Ubi-α−specR22S] α−speclm111 α−specRG41 P[Ubi-α−specR22S] Downloaded from http://www.jbc.org/ by guest on November 12, 2014 + ; Name used in paper (selection criteria) One copy rescue (1xP[w+], Cy Hu+) Two copy rescue (2xP[w+], Cy+ Hu+) One copy coexpression (1xP[w+], Cy Hu) One copy coexpression (1xP[w+], Cy Hu) Two copy coexpession (2xP[w+], Cy+ Hu) Two copy rescue (2xP[w+], Cy Hu+) α−speclm111 Three copy rescue (3xP[w+], Cy+ Hu+) P[Ubi-α−specR22S] CyO, Cy ; α−speclm111 TM6B, Tb Hu e One copy coexpression (1xP[w+], Cy Hu) P[Ubi-α−specR22S] P[Ubi-α−specR22S] ; α−speclm111 TM6B, Tb Hu e Two copy coexpression (2xP[w+], Cy+ Hu) α−specRG41 P[Ubi-α−specR22S] Two copy coexpression (2xP[w+], Cy Hu) P[Ubi-α−specR22S] ; CyO, Cy TM6B, Tb Hu e P[Ubi-α−specR22S] α−specRG41 P[Ubi-α−specR22S] P[Ubi-α−specR22S] 18 ; TM6B, Tb Hu e Three copy coexpession (3xP[w+], Cy+ Hu) Drosophila development without spectrin tetramerization Figure 4 S 22 ue αResc r S 22 xp αR oe c S 22 ue αR sc re S 22 xp αRcoe pe -ty ild w A 0 1 1 2 _ + + + _ + + 2 S g 22uin αResc r protein detected # transgenes αR22S Total α protein present αR22S wt α + B Total α-spectrin C α-spectrinR22S 19 + _ Downloaded from http://www.jbc.org/ by guest on November 12, 2014 Tub Drosophila development without spectrin tetramerization Figure 5 1cx 22oC A’ A’’ B’ B’’ * Dlg B DNA 1cx 29oC * Dlg DNA C’ C C’’ 1cr 22oC * Dlg DNA D’ D D’’ 1cr 29oC * DNA α-spec 20 Downloaded from http://www.jbc.org/ by guest on November 12, 2014 A Drosophila development without spectrin tetramerization Figure 6 A B wt 1cr F H’ H wt K HRP B’’ C 2cr 3cr 2cx F’ 3cr H’’ SYT F’’ I α-sp 1cr L 3cr wt F-actin 1cr * D D’ 3cx αwt α1cx G M * 21 2cx α-sp SYT * F-actin 1cr G’’ J * * α OE 2cx I’’ SYT E G’ 2cx I’ HRP * C’ * F-actin Downloaded from http://www.jbc.org/ by guest on November 12, 2014 2cr B’
© Copyright 2024