Pupal squashes were performed in approximately 6-hour intervals starting at 24 h ASP (see Methods 24). The typical cell distribution and cell morphology obtained with the squash technique for the period from 30 hours to 48 hours ASP is presented in Figure 1 panels A, C and E. The corresponding immunofluorescence indicates that cells immunostaining for muscle proteins are large rounded cells (myosin, Z(210) and projectin in panels B, D and F respectively). This pattern is similar to the images obtained from frozen cryostat sections of pupae at the same stage (data not shown). Because many muscles besides IFMs are also forming at the same time, the identity of specific cells as early IFM myotubes was evaluated by immunolabeling for the Z(210) protein, which is only found in IFMs and the large cells of the Tergal depressor of trochanter (TDT or jump muscles) 3. Cells expressing Z(210) were identified as early as 40 hours ASP. Many, but clearly not all, cells within a pupal squash stain positive for the Z(210) antibody (compare Figure 1C and 1D and arrows indicating Z(210) negative cells). As seen at higher magnification, the cells staining for the Z(210) protein are also multinucleated as shown by DAPI staining, (Figure 1G and 1I). Because of both the presence of the Z(210) protein and the multinucleated pattern, we consider these early cells as precursors for the IFMs or the large cells of the TDT.

At each stage tested, the presence and pattern for the following myofibrillar proteins were assessed: projectin, myosin heavy chain, α-actinin, kettin, Z(210) 3 and F-actin. Staining with projectin antibodies appears, by 30 hours, as few randomly dispersed aggregates throughout the cells' cytoplasm (data not shown). By 36–40 hours ASP, the projectin staining becomes brighter and is found within a greater number of aggregates (Figure 2A). The identity of the cells showing projectin in this aggregate pattern as early IFM myotubes was evaluated by immunolabeling for the Z(210) protein. As shown in Figure 2B, the cells where projectin is found in an aggregate pattern also show immunostaining for Z(210) which colocalizes over the same aggregates. Double immunofluorescence staining, between projectin and either α-actinin or kettin demonstrates that staining for the other Z-band-associated proteins can be aligned over the projectin's aggregates (Compare Figure 2E and 2F for projectin and kettin). Colocalization of other Z band proteins with the Z(210) protein was also obtained (Figure 2G and 2H for α-actinin and Z(210) respectively), again identifying these myotubes as the precursors of the IFMs or large cells of the TDT. The staining for F-actin obtained with FITC-labeled phalloidin shows that actin also colocalizes with these aggregates (compare Figure 2C and 2D for projectin and F-actin) consistent with the early association of growing thin filaments with the forming I-Z-I complex, which has been reported before 21. These results are, therefore, consistent with the early anchoring of projectin to the I-Z-I complex during its assembly within the forming myofibrils.

For each protein tested, the same immunofluorescence pattern was observed when the different antibodies available against a particular protein were used (see Methods for list of antibodies). That the colocalizations are not an artifact of bleed through of the secondary antibodies between the two filters is supported by the data from single immunofluorescence where the aggregate pattern was observed individually for all proteins tested (data not shown). Also, when FITC conjugated secondary antibody is used alone, there is no fluorescence in the rhodamine channel and vice-versa and staining with either of the secondary antibodies alone is always negative (data not shown).

By 48 hours ASP, the staining for projectin, as well as kettin, α-actinin and Z(210) has changed to a pattern of ordered bands where all tested Z-band-associated proteins are colocalized (compare Figure 3A and 3B for projectin and α-actinin respectively). The identity of these cells as IFM was ensured by double staining with Z(210) as shown in Figure 3C and 3D (projectin and Z(210) respectively), as well as 3E and 3F (α-actinin and Z(210) respectively).

The myosin staining was also evaluated in these two pupal stages and compared to the aggregate pattern obtained for the Z-band-associated proteins. At 40 hours ASP, myosin staining appears diffuse throughout the entire cytoplasm (Figure 4B). Double staining between projectin and myosin shows the presence in the same cells of the aggregate staining for projectin and a diffuse cytoplasmic distribution for myosin (compare Figure 4A and 4B). Even at a later stage (48 hours ASP), myosin is still found in a diffuse, random pattern with a stronger signal located around the plasma membrane (Figure 4D) whereas the double immunofluorescence with projectin shows the I-Z-I band localization (Figure 4C and 4D).

By 60 hours ASP, a fully organized striated pattern is recognizable for all the tested proteins including myosin (data not shown).

Pupal squashes of several mutant stocks were performed at two different times during pupal development: approximately 40 hours and 48 hours asp. At both stages the presence and localization of several myofibrillar proteins were assessed as described above and compared to the wild type assembly pattern. These two stages were specifically chosen as they represent respectively the time point when aggregates are the most abundant and easier to visualize and the time point for early Z-band assembly as described above.

All three myosin mutations, Mhc⁷, Mhc¹⁰ and Mhc¹¹, are IFM-specific null mutations and there is no staining with any anti-myosin antibody available to us, even when the images were purposefully overexposed (Figure 5B). In the same myosin-negative cell, projectin clearly shows a regular banding pattern (Figure 5A) similar to the one observed in wild type pupae (Figure 3A for example). As shown in Figure 5 for the Mhc⁷ allele, F-actin and projectin assemble following a pattern similar to the wild type, first as aggregates around 40 hour ASP (Figures 5C and 5D for projectin and F-actin respectively) and then as regularly spaced Z-bands after 48 hours ASP (Figures 5E and 5F for projectin and F-actin respectively). α-actinin and kettin also assemble following the wild type pattern (data not shown). The assembly data obtained for the Mhc¹⁰ and Mhc¹¹ alleles are identical to the data presented in Figure 5 for Mhc⁷ (data not shown).

Two actin mutations were also selected, KM88 and E93K, which are both mutant alleles of the IFM-specific Act88F gene 2526. In the E93K mutant, actin still assembles in thin filaments but in the adult IFMs, the Z-bands are degenerated 26. In this mutant allele, projectin assembles properly through the expected steps. At the 40-hour time point co-staining over aggregates is visible between projectin and actin (Figure 6A and 6B), as well as with kettin and α -actinin (data not shown). The staining changes by 48 hours ASP toward a Z-band pattern indistinguishable from the pupal wild type (Figure 6C and 6D). Therefore, the E93K actin mutation does not disrupt the early formation of Z-bands and the assembly of projectin during pupation. In adult E93K flies, IFM myofibrils can be prepared and immunofluorescence staining indicates that projectin is still present within the myofibrils (Figure 6E). By comparison to the immunostaining obtained for myosin (FITC/green staining in Figure 6F), projectin's localization over the I-Z-I region is further confirmed, as the two fluorescence patterns do not overlap. Therefore, even though Z-bands are degenerated in the E93K adult IFM myofibrils, the localization of projectin over the I-Z-I region is maintained.

In KM88 mutant flies, no actin is present in the IFMs, and in the adults both the thin filaments and the Z bands are absent 2527. As shown in Figure 6G for projectin, the early step involving the formation of the aggregates takes place in the KM88 mutants, whereas F-actin is not detectable even with an overexposed image (Figure 6H). The early colocalization is also observed for kettin (Figure 6I) and α-actinin (data not shown). The identity of these cells as IFM precursors was confirmed by the staining with an anti-Z(210) antibody, such that Z(210)-positive cells show aggregates but are negative for actin staining (data not shown). So, even in the absence of thin filaments, early aggregation of Z-band associated proteins takes place, and projectin is recruited within these aggregates. The next step involving the formation of early Z-bands is, however, not observed. In late pupae (50 hours and older) a striated pattern can be observed in some fibers under the phase contrast setting (Figure 6J). As shown in Figure 6K and 6L, these same muscles do not exhibit a recognizable striated Z-band pattern when stained with either projectin or kettin. Therefore, the final Z-band assembly for projectin, kettin and α-actinin does not take place in the KM88 mutant. Since IFM myofibrils cannot be prepared from adult KM88, we were not able to further assess by immunofluorescence the presence and localization of projectin in adult IFMs.

Lakey et al. 28 demonstrated that, when purified IFM myofibrils are incubated in the presence of the protease, calpain, the Z-bands are preferentially digested as shown by the specific loss of α-actinin and kettin (see also Figure 7). We performed a similar analysis to assess the behavior of projectin in response to calpain treatment and its attachment sites within the myofibrils.

Calpain-digested myofibrils were analyzed by immunofluorescence microscopy as described in Methods. Non-treated myofibrils were incubated in calpain activation buffer but without calpain for the same amount of time and condition as the treated myofibrils. When no calpain was added, all proteins tested (kettin, α-actinin, actin, myosin and projectin) are detected within their respective unchanged sarcomeric domains (Figure 7 panels A, C and E for kettin, myosin and F-actin respectively; Figure 7 panels G, I and K for projectin). Upon addition of calpain, all staining is lost for both kettin (Figure 7, Panel B) and α-actinin (not shown), and this result is consistent with previously published data 28. Myofibrils, however, could still be detected under the phase contrast setting indicating that myofibrils devoid of Z bands are still present (not shown). Accordingly, A-band staining using a myosin antibody was still observed in calpain-treated myofibrils (Figure 7D). Similarly, F-actin visualization using FITC-labeled phalloidin staining does not give indication of any visible loss of F-actin upon calpain digestion (Figure 7F). Therefore, the data indicate that under the conditions used, detectable calpain digestion is limited to the Z band and that thin and thick filaments remain relatively untouched and in position. Another difference observed sometimes between the no calpain and the calpain-treated slides was that myofibrils tended to be smaller after calpain treatment (Figure 7C and 7D). These data were usually obtained using the protocol with a centrifugation step after the calpain treatment (see Methods). Later in the analysis, this step was omitted, and no significant decrease in myofibril length was observed after digestion. It is probable that the fibrils are more fragile after the calpain treatment and, therefore, tend to break apart more easily during the centrifugation step.

Calpain-digested myofibrils were then analyzed using five different projectin antibodies, namely MAC150, P5, Core1P, 3b11 and kinase (see Methods for description and origin of these antibodies). All preparations were double stained with an anti-kettin antibody to insure that calpain digestion has taken place (not shown). Projectin staining after calpain treatment is still present when projectin is detected using any of the five antibodies, as presented in Figure 7G,7H,7I,7J,7K,7L for P5, Core1P and 3b11 respectively. This indicates that the projectin molecule is not being released from the adult IFM myofibrils upon digestion of the Z-band. So, even at a concentration when all other tested Z-band proteins have been removed as shown by the immunofluorescence data and by gel analysis (data not shown), projectin is still within the myofibrils at its proper localization. These data are consistent with the existence of protein-protein interactions between projectin and the thick and/or thin filaments within the mature myofibrils, insuring physical integrity and strength.

Pupae were staged according to the protocol of Bainbridges and Bownes 24. Pupae were collected at the immobile white prepupae stage with everted spiracles. The pupal case becomes brown within an hour of immobility and marks the beginning of pupation 24. The pupae were then aged for the appropriate time at 22°C, and the timing used in this study is based on hours after the start of pupation (abbreviated as ASP). Developmental landmarks were also used to confirm staging 24. Because of the asynchrony of pupal development, the chronology indicated in this paper represents the times/stages where any particular pattern is predominant. Staged pupae were hand-dissected from their pupal case using fine glass needles. Either the whole pupa or the thoracic area (for pupae aged 40 hours ASP and later stages) was used and the tissues were teased apart. The tissues were spread on poly-L-lysine coated slides by applying hand pressure with a coverslip and processed for immunofluorescence as described below.

Three myosin mutants were obtained from Dr. Charles Emerson's laboratory (U. Penn. Medical School): Mhc⁷, Mhc¹⁰ and Mhc¹¹. All three are null mutations, which specifically disrupt the myosin heavy chain protein only in IFMs. These stocks are, therefore, homozygous viable, though flightless. The two actin mutations, E93K and KM88, were obtained from Dr. John Sparrow's laboratory (York University, UK). They are IFM-specific as they are mutated alleles of the gene coding for the IFM-specific actin isoform (Act88F), and are homozygous viable flightless.

Myofibrils were extracted from adult wild type Oregon R or from E93K flies using a procedure, which yields primarily asynchronous myofibrils 3. Approximately 0.5 grams of flies were homogenized in Hodges/EGTA buffer (0.1 M KCl, 1 mM MgCl₂, 2.5 mM EGTA, pH 7) containing the following protease inhibitors: 0.5 mM phenylmethanesulfonylfluoride (PMSF), 0.1 mg/ml soybean trypsin inhibitor (STI), 5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 0.2 mg o-phenanthroline (OPT). This mixture was filtered and submitted to several sucrose gradient centrifugations as described 3. In the last gradient, the band at the 1.25 M/2.5 M sucrose interface was collected as this fraction is highly enriched in IFM myofibrils. The preparation was kept at 4°C for no more than a week before being used for immunofluorescence.

Myofibrils were digested with calpain using the procedure of Lakey et al. 28. Myofibrils were typically washed once with Hodges/EGTA buffer to remove the sucrose. Pelleted myofibrils were further washed 2 times before being resuspended in calpain activating solution (CAS: 0.1 M NaCl, 20 mM Tris-HCl pH 7, 2 mM MgCl₂, 10 mM β-mercaptoethanol, 2 mM NaN₃, 5 mM CaCl₂). In early studies, myofibrils were treated with calpain in solution, and then pelleted by centrifugation before being deposited on a slide for analysis. In later studies, a drop of untreated fibrils was first deposited on poly-L-lysine coated slides and left to settle for approximately 10 minutes to facilitate myofibril absorption to the slide. Various amounts of calpain (Sigma) in calpain activating solution were then added directly onto the myofibrils. In both protocols, calpain amounts typically ranged from 0 μg (in controls) to 72 μg of calpain per slide. After addition of the calpain, myofibrils were incubated at 25°C for 90 minutes. Calpain-treated myofibrils were then fixed with 4% formaldehyde in 1X PBS immediately at the end of the incubation period and processed for immunofluorescence microscopy as described below.

The immunofluorescence procedure was as described before 7. For double labeling, the two primary antibodies were applied together, as well as, the two secondaries. Primary antibody combinations were selected such that each primary would be recognized by a different secondary (see below). Both rhodamine and fluorescein tagged-secondary antibodies (Pierce) were used for identification at a 1:200 dilution. F-actin was detected using FITC-labeled phalloidin (Sigma) at a dilution of 1:100, which was routinely added with the secondary antibody. Slides were mounted in mounting medium with DAPI (Vectorshield Inc.). In all control immunofluorescence experiments, reacting the preparations with the secondary antibodies alone indicated no background staining. Slides were examined by epifluorescence microscopy at either 80× or 200× magnification. Early images were recorded on Kodachrome color slides, which were subsequently digitized using a scanner. In later stages, fluorescent images were directly recorded using a microscope-mounted digital camera. All images were processed and figures assembled using Abobe Photoshop^R software.

A cDNA fragment representing a portion of the central core region of the projectin protein 13 with 2 Ig and 6 Fn₃ domains was amplified by RT-PCR. The DNA fragment was then gel-purified and subcloned into a pQE vector (Qiagen Inc.), which provides an NH₂-terminal 6X His tag. The bacterial fusion protein was induced and purified on Ni-NTA columns from bacterial cultures, as recommended by the manufacturer (Qiagen Inc.). The Core1P purified fusion protein was run on SDS-polyacrylamide gels followed by transfer onto nitrocellulose membranes. The area of the membrane containing the protein was cut out and used for rabbit immunization (Pocono Rabbit Farm). The Core1P antibody specificity was assessed by standard Western analysis of total adult proteins as described before 7 (data not shown).

The following antibodies were used in the immunofluorescence studies.

Projectin: MAC150, Core1P, 3b11, P5 and kinase; Myosin heavy chain: 3e8 and 722; α-actinin: 4g6 and MAC276; Kettin: MAC155; Z(210): 3d10. The three "MAC" antibodies are rat monoclonals (generous gifts from Dr. Belinda Bullard, EMBL, Heidelberg, 4). Antibodies 3e8, 4g6, 3b11, P5 and 3d10 are mouse monoclonals (generous gifts from Dr. Judith Saide, Boston University School of Medicine, 3). 722 is a rabbit polyclonal (a generous gift from Dr. Dan Kiehart, 43). Kinase is a rabbit polyclonal directed against twitchin kinase, which cross-reacts with projectin kinase domain (generous gift of Dr. G. Benian, Emory University). Core1P antibody is described above.