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Filamin protects myofibrils from contractile damage through changes in its mechanosensory region [1]

['Lucas A. B. Fisher', 'Department Of Biology', 'Mcgill University', 'Montreal', 'Quebec', 'Belén Carriquí-Madroñal', 'Interfaculty Institute Of Cell Biology', 'Universität Tübingen', 'Tübingen', 'Tiara Mulder']

Date: 2024-07 

Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.

Muscles work by contracting and relaxing in response to signals from the nervous system. Muscles are made up of long, slender cells called muscle fibers. Inside these fibers are even smaller units called sarcomeres, which are responsible for the actual contraction of the muscle. The sliding of actin and myosin filaments shortens the sarcomeres, causing the muscles to contract. As sarcomeres contract, the entire muscle shortens, pulling on the tendons attached to it. This pulling action is what creates movement. Sarcomeres however are prone to contractile damage and so they have proteins to hold the sarcomere together. One of these proteins is filamin, a large protein which has a special elastic part called the mechanosensitive region, which might be crucial for providing stability to the sarcomeres as they contract. To test this hypothesis, we made versions of filamin with increased or decreased elasticity and studied how muscles behaved with these changes. We find that when filamin lost its elasticity, sarcomeres are more prone to breaking while when filamin has extra elasticity, the sarcomeres do not break and instead form large protein aggregates which we think represent the sarcomere response to contractile damage. This research gives us a better understanding of how our muscles sustain contractile damage, which is important for everyone from athletes to people recovering from injuries.

Funding: This work was supported by operating grants MOP-142475 to FS and PJT-155995 to FS from the Canadian Institutes of Health Research and by RGPIN-02984-2022 to NGM from the Natural Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we use novel filamin mutants to analyze the effect of the MSR on myofibril ultrastructure. We focused on the indirect flight muscles (IFM) because of their regular sarcomere pattern [ 25 , 29 , 30 ]. We found that locking the MSR of filamin into a closed state leads to Z-disc rupturing while locking the MSR into an open state leads to enlarged Z-discs, which we interpret as a compensatory mechanism for the sarcomere damage that occurs during contractions to stabilize the Z-disc and prevent it from breaking. We found that the removal of the entire MSR region leads to a ruptured disc and myofibril disintegration. We found that the Ig domain pairs 16–17 and 18–19 are required for Z-disc stability. We found that filamin dimerization is also required for Z-disc cohesion. Finally, we found that muscle contraction is required for myofibril rupture in filamin mutants. Our data provides a detailed analysis of the domains of filamin involved in myofibril stability and damage sensing, and we provide new tools to study filamin in Drosophila.

Filamin is sensitive to mechanical pulling forces [ 26 ], which makes it ideal for sensing Z-disc stretching damage and initiating repair signals. The mechanosensitive region (MSR) of Drosophila filamin consists of the Ig domains 14–19, which adopts a globular structure ( Fig 1C ; [ 27 ]). The MSR is organized into three Ig domain pairs, in Drosophila these are Ig 14–15, 16–17, and 18–19. Interaction sites between the contact faces of Ig 16 and 17, and another one between Ig 18 and 19 keep the MSR in a closed inactive state [ 28 ]. However, each of these interactions is sensitive to mechanical pulling forces and upon pulling forces of 3–5 pN, the intradomain interactions are lost and the Ig domains are free to interact with other proteins [ 14 , 26 , 28 ]. The MSR is in a closed inactive state when its domain pairs are tightly bound or in an open active state when its domain pairs are free to bind interacting proteins.

A) A transmission electron microscope image of a sarcomere; the Z-discs are highlighted in magenta, and arrows point towards the direction in which actin filaments are pulled during the contraction cycle. B) Cartoon model of filamin at the Z-disc. The dimerization domain at the C-terminus points away from the Z-disc. C) AlphaFold model of the mechanosensitive region of Drosophila filamin (AFQ9VEN1). D-G) Confocal microscopy images of different filamin mutant conditions. Filamin alleles were analyzed over the Df(3R)Ex6176 deletion mutant. Actin filaments are shown in green and Zasp52-mCherry in magenta. Individual channels are shown in grayscale. D) filamin WT does not have any obvious muscle defect. E) filamin WT-GFP also does not have any obvious muscle defect, suggesting that the C-terminal GFP tag does not affect filamin function. F) In filamin closed-GFP mutants, the myofibril appears ruptured at the Z-disc, marked with Zasp52-mCherry (red arrows). A slight increase in Zasp52 levels occurs at the breaking points. G) In filamin open-GFP mutants the myofibrils are not ruptured but have occasional enlarged Z-discs (purple arrows). The scale bar is 5 μm. H-J) Flight percentage plots for homozygotes at 1 week (H) and 2 weeks (I), as well as for heterozygotes at 2 weeks (J). In panels H-J, N = 30 flies for each data bar.

Filamin is a large protein consisting of an elongated structure with a length of approximately 80 nm [ 13 , 14 ] Human filamins are composed of two identical subunits, each of which contains an N-terminal actin-binding region composed of two Calponin Homology (CH) domains, followed by 24 immunoglobulin-like (Ig) domains, the last Ig domain being a dimerization domain [ 13 ]. The Ig domains are arranged into a flexible rod-like structure that allows filamin to interact with a wide variety of binding partners, including membrane receptors, signaling proteins, and other cytoskeletal proteins. Humans have three filamin genes Filamin-a, Filamin-b, and Filamin-c. Structurally, Filamin-a, -b, and -c are highly similar, but their expression patterns vary. Filamin-a is the most abundant and widely distributed member [ 13 ]. Filamin-b shows ubiquitous expression with elevated levels in endothelial cells and chondrocytes [ 15 ]. Filamin-c is primarily expressed in adult muscles [ 13 , 16 ]. Drosophila has a single filamin gene called cheerio that replaces the three vertebrate filamins [ 17 ]. Here we refer to cheerio as filamin. The structure of Drosophila filamin is identical to the vertebrate filamins with the exception that is has only 22 Ig domains instead of 24. In muscles, filamin localizes to the Z-disc and to the myotendinous junctions in both vertebrates and Drosophila [ 18 – 21 ]. Filamin is organized into the Z-disc with its actin-binding domains at the center of the Z-disc, where they bind actin filaments from opposing sarcomeres, and the dimerization domain at the periphery of the Z-disc ( Fig 1A and 1B ; [ 22 , 23 ]). This organization is parallel to the contractile forces of muscles. In cells, actin filaments are crosslinked at variable angles from 35 to 90 degrees [ 24 ]. In contrast, at the Z-discs actin filaments are parallel to each other and filamin connects the actin filaments from two opposing sarcomeres [ 25 ].

Concentric contractions occur when the muscle fibers shorten in response to a load or resistance. Eccentric contractions occur when the muscle fibers lengthen while still under tension. Eccentric contractions generate more force than concentric contractions but cause myofibril damage [ 6 ]. The eccentric myofibril damage is characterized by a series of structural steps that start with the overstretching and breaking of the weakest Z-disc in the myofibril [ 7 ]. Then extensive Z-disc remodeling follows including the widening of the Z-disc [ 8 ]. Finally, the Z-disc structure is reassembled. During this process, some Z-disc proteins, including Filamin, Xin, Hsp70, and αB-crystallin [ 9 – 11 ], accumulate at the lesions and are thought to mediate Z-disc stability and repair [ 9 – 11 ]. Filamin is one of the earliest proteins to localize at damaged regions and a commonly used marker of myofibrillar damage [ 10 , 12 ].

Striated muscles are big cells that in addition to the typical cellular constituents are characterized by large cytoskeletal cables called myofibrils which are composed of repeating subunits called sarcomeres [ 1 ]. Sarcomeres are responsible for the contraction of muscles and are made up of actin and myosin filaments. The Z-disc is a thin structure that defines the boundaries of the sarcomere and serves as the anchor point for actin filaments [ 2 ]. The M-line is at the center of the sarcomere where myosin filaments are anchored [ 2 ]. Myosin heads slide actin filaments towards the center shortening the sarcomere to provide the basis for muscle contraction [ 3 ]. The Z-disc is also where the myofibril diameter is set [ 4 ], and it is a hub for metabolic enzymes [ 5 ].

One-week-old flies were anesthetized on an ice pad. Then a 10 μl pipette tip was glued to their thoraces using fast-drying transparent nail polish. The flies were placed in the middle of the sensor using a 3D printable micromanipulator [ 37 ]. Flies were left to recover for 5 minutes at room temperature. After careful placement of the flies in the middle of the two detectors, we recorded the reflected light from both wings for periods of 30 seconds. Flies were stimulated to start flight by directing a gentle puff of air at them. Plots and correlation coefficients were calculated using custom R scripts. The double-wing sensor is composed of two QRD1114 sensors (DigiKey QRD1114) connected to an Arduino UNO microcontroller. The two QRD1114 sensors are mounted into a custom-made 3D-printed holder. The maximum reflectance signal occurs when the wing is 0.635 mm from the sensor. 3 mm is the maximum range at which the sensors detect something. The wing movement was recorded in tethered flies. The data was analyzed in R software using custom scripts. Briefly, the output of the Arduino was saved as a CSV file containing two columns, one per wing. We selected a 5-millisecond time window randomly, plotted the results, and calculated the Pearson correlation value of the wing movements. We repeated the process 50 times and counted the number of synchronous and asynchronous events. We analyzed 10 flies per genotype and recorded 1 minute of continuous flying. We used a threshold value of r > 0.5 to classify synchronized and unsynchronized flying events. S3 Data contains supporting information for myofibril phenotype data, wing beat synchronization data, and GFP intensity data. For flight assays, 30 flies were released individually from a plastic vial: if they flew upwards, they were marked as a flyer, whereas if they fell or glided to the ground they were marked as a non-flyer.

Newly eclosed flies were placed in a custom-made immobilization chamber with a diameter of 6.35 mm and a length of 5 cm. In this chamber, flies were able to walk along the chamber but had limited room, which prevented flight. After 13–15 days in the chamber at 25°C their IFM were dissected and imaged. Simultaneously, flies of the same genotype were kept in normal vials as the flying group.

Dissection and microscopy experiments were done following our previous protocol [ 5 , 31 , 34 – 36 ]. Briefly, thoraces from female flies were cut in half longitudinally using a sharp blade, then fixed for 1 h at room temperature using 4% formaldehyde. Following three washes with PBS triton 0.1%, the muscles were further dissected to remove them from the thoracic cuticle. The isolated muscles were then incubated in PBS with 1:1000 488-Phalloidin or 555-Phalloidin (Acti-stain, Cytoskeleton Inc.) to stain the actin filaments. After washing with PBS triton 0.1% again, the muscles were mounted in Mowiol 4–88 mounting media (Sigma 9002-89-5). To image the muscle ultrastructure, we used a Leica TCS SP8 Confocal Microscope. Images were taken with an HC PL APO 63x oil NA = 1.4 objective. We used a 488 nm, 20 mW, AOTF laser for GFP and 488-Phalloidin and a 552 nm, 20 mW laser for mCherry and 555-phalloidin. Only the outer layer of myofibrils was used because phalloidin does not penetrate well into the tissue. Imaging settings were kept identical within experiments. Well-stained muscles were selected and aligned using the 63X objective without any additional digital zoom. A large area at the center of the muscles was selected. Then, randomly selected areas within this area were imaged at 9X digital zoom with a pixel resolution of 1024x1024. At least 10 muscles were analyzed per condition. Finally, we cropped an area of the scanned image to show the representative phenotypes. To calculate the ratios of affected myofibrils, we used the uncropped images that typically contain 10–15 myofibrils. The ratio of affected myofibrils is the number of affected myofibrils in an image divided by the total number of myofibrils in the same image. S1 Data contains a summary table of myofibril phenotypes and S3 Data contains the myofibril counts. To measure GFP intensity, we utilized the following ImageJ script, which creates a 250-pixel circle around a selected spot and calculates the average gray value.

Western blotting experiments from adult flies were done as previously [ 30 ]. Briefly, 20 adult flies were homogenized in 100 μl of 2x SDS running buffer via mortar and pestle, boiled at 100°C for 10 minutes, spun down, and then the supernatant with all the soluble proteins was collected. The soluble fraction was run on a NuPage (Thermo Fisher) Tris-Acetate gel and transferred onto a nitrocellulose membrane. Rabbit anti-GFP antibody (Chromotek) was then added at a 1:5000 dilution concentration, followed by anti-Rabbit ECL (Millipore) at 1:10000 concentration. Membranes were scanned with a C-DiGit Blot Scanner (Licor). As loading control, we used Ponceau staining (5% glacial acetic acid and 0.1% Ponceau S), followed by washing with 1X Tris-Buffered Saline 0.1% Tween.

The deletion mutants are derived from pGE-attBGMR-cher WT-GFP , which contains the last five exons of cher cloned from BAC RP98-2L16 with a C-terminal mGFP6 tag cloned into pGE-attB-GMR (DGRC Stock 1295; RRID: DGRC_1295) via EcoRI and KpnI sites [ 27 ]. DNA fragments lacking specific domains were generated by overlap extension PCR and subcloned by conventional cloning into pGE-attB-GMR-cher240WT-GFP replacing wild type sequences. The resulting plasmids were sequence-verified and integrated into the attP site of cher s24-attp . The correct integration was confirmed by PCR; the w+ marker was removed using Cre recombinase [ 33 ]. The aligned amino acid sequences of all mutant forms used in this study are available as a supplementary document; proteins were aligned using the ClustalW function from MacVector (version 18.0.2). The transcription start site for the cher90 isoform has been omitted from the rescue construct, resulting in the absence of the small cher90 isoform. Since GFP is positioned at the C-terminus, all other isoforms are tagged.

Drosophila stocks were cultured and maintained on standard cornmeal glucose media. Zasp52-mCherry is a gene trap made by swapping the Zasp52 MI02988 MIMIC transposon with an artificial exon containing the mCherry coding sequence [ 4 , 31 ]. Cher s24-attp contains an attp landing site, is on a white mutant background, and is used as the founder line for all the cher knock-in mutants [ 27 ]. The replacement alleles cher WT , cher WT-GFP , cher open-GFP , cher closed-GFP cher ΔIg14-21-GFP were described previously [ 27 ]. The Df(3R)Ex6176 deficiency uncovers the filamin gene and was generated through FLP-induced recombination between two FRT-carrying transgenic insertions at 3R:17,053,662 and 17,149,107 [ 32 ]. S1 Data contains a list of the full genotypes that were used in the main figures.

Results

Genomic engineering a set of small deletion mutants tagged with GFP To reveal how Drosophila filamin mediates its function during muscle maintenance, we performed a structure-function analysis of the mechanosensitive C-terminus. We generated 7 new lines (Fig 3A and 3B, and S2 Data), each with a small deletion that removes specific Ig domains of the C-terminus, using a knock in approach [27]. We also included in our analysis filaminΔIg14-19-GFP that lacks the entire MSR [27]. Western blot analysis of these filamin forms showed that each line expressed a filamin protein of the expected size (Figs 3C and S2). The resulting constructs were verified by sequencing and are incorporated into the filamin gene using filamins24-attp as the landing site. We made 8 mutants: filaminΔIg14-22-GFP, filaminΔIg14-21-GFP, filaminΔIg15-21-GFP, filaminΔIg16-17-GFP, filaminΔIg18-19-GFP, filaminΔIg20-21-GFP, and filaminΔIg22-GFP. The transcription start site for the cher90 isoform has been omitted from the rescue construct, resulting in the absence of the small cher90 isoform [27]. Since GFP is positioned at the C-terminus, all other isoforms are tagged. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Generation of novel filamin alleles tagged with a C-terminal GFP. A) Cartoon of the cheerio/filamin locus. Boxes represent exons. The red boxes denote the exons that encode the MSR. The filamins24 mutant is a deletion of the C-terminal part of the filamin gene replaced with an attB Integrase landing site (gray line). B) Illustration of the deletion mutants generated. The MSR is colored in red and Ig domains 20–22 in purple The rows in descending order illustrate filaminWT-GFP, filaminΔIg14-22-GFP, filaminΔIg14-21-GFP, filaminΔIg15-21-GFP, filaminΔIg16-17-GFP, filaminΔIg18-19-GFP, filaminΔIg20-21-GFP, filaminΔIg22-GFP, and filaminΔIg14-19-GFP, which was generated previously [27]. C) Western blots showing the molecular weights of the truncated filamin proteins using an anti-GFP antibody from thorax lysates. All the deletions have the correct predicted molecular weights, filaminΔIg15-21-GFP 175 kDa, filaminΔIg14-21-GFP 185 kDa, filaminΔIg14-22-GFP 197 kDa, filaminΔIg22-GFP 257 kDa, filaminΔIg20-21-GFP 247 kDa, filaminΔIg18-19-GFP 248 kDa, filaminΔIg16-17-GFP 247 kDa, filaminWT-GFP 267 kDa. D) Cartoon of the Ig domains 14–22. The MSR is colored in red and the following Ig domains 20–22 in purple. The interactions between the Ig domains 14 with 15, 16 with 17, and 18 with 19 are the structural basis of the MSR function. https://doi.org/10.1371/journal.pgen.1011101.g003

Filamin dimerization is required to prevent Z-disc rupturing To crosslink actin filaments, actin-binding proteins such as filamin must have at least two actin-binding domains. A single filamin monomer has only one actin-binding domain and is not able to crosslink actin filaments. The filaminΔIg22-GFP allele removes specifically the dimerization domain and allows us to interrogate the requirement of forming filamin dimers. The sarcomeres of the filaminΔIg22-GFP mutant muscles exhibited clear ruptured Z-discs, although no fraying of the myofibrils was observed (Fig 5A and 5B). We then investigated whether preventing muscle contractions would ameliorate the filaminΔIg22-GFP mutant phenotype. Similarly to filaminclosed-GFP flies, the ruptured phenotype of the filaminΔIg22-GFP was rescued by inhibiting flight (S3C Fig). Overall, these findings suggest that dimerization is necessary to protect the Z-disc from contractile damage, but not required to prevent actin filaments fraying from the myofibril. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Mutants lacking one pair of Ig domains, or the dimerization domain have distinct myofibril phenotypes. A-E) Confocal microscopy images of diverse filamin mutant muscles. Filamin alleles were analyzed over the Df(3R)Ex6176 deletion mutant. Actin filaments are shown in green and Zasp52-mCherry in magenta. A) Example of normal myofibrils. B) Example of ruptured myofibrils present in the dimerization domain mutant filaminΔ22-GFP (red arrows). C) Example of ruptured myofibrils present in the filaminΔIg16-17-GFP mutant (red arrows). D) Example of ruptured myofibrils present in the filaminΔIg18-19-GFP mutant (red arrows). E) Myofibrils with enlarged Z-discs present in the filaminΔ20-21-GFP mutant. In this mutant, the myofibrils do not break. F) Plot of the ratio of ruptured myofibrils over the total number of myofibrils. All single-domain pair mutants have ruptured myofibrils except for the filaminΔ20-21-GFP mutant. G) Plot of the ratio of enlarged Z-discs over the total number of myofibrils. Only the filaminΔ20-21-GFP mutant has enlarged Z-discs. H) Plot of the ratio of frayed myofibrils. Confidence intervals were calculated at 95 by an exact binomial test, and the p-values using a 2-sample test for equality of proportions with continuity correction. https://doi.org/10.1371/journal.pgen.1011101.g005

Within the MSR, both mechanosensitive dimers contribute to mechanosensing and filamin lacking Ig 20–21 behaves like filamin open-GFP We then analyzed the muscle phenotypes of three small deletions that remove single-domain pairs, filamin Ig domains 16–17 and Ig 18–19, which are part of the MSR, and Ig domains 20–21, which are just outside the MSR (Fig 3B and 3D). Muscles from the filaminΔIg16-17-GFP and the filaminΔIg18-19-GFP mutants had ruptured myofibrils (Fig 5C and 5D). Overall, these mutants behave as filamin closed-GFP mutants (Figs 1 and 2). Unlike all the other deletion mutants, the muscles from the filaminΔIg20-21-GFP mutant flies have enlarged Z-discs, like the ones observed in filamin open-GFP (Fig 5E). We measured the size of the enlarged Z-discs using Zasp52-mCherry in the two mutants and noticed that they have identical sizes (S4 Fig). Actin accumulates at very high levels in the enlarged Z-discs and covers a slightly wider area compared to Zasp52 (5E Fig). However, we noted that unlike in filamin open-GFP mutants, the filaminΔIg20-21-GFP homozygote mutants can fly, suggesting that the flightless defect observed in filamin open-GFP mutants is not linked to the enlarged Z-discs. Finally, we counted the number of ruptured and enlarged myofibrils in all the small deletion mutants and noted that the mutants would either have ruptured myofibrils or enlarged Z-discs but not both (Fig 5F and 5G), which is identical to what we observed in the open and closed mutants. In contrast to the large mutants, the single-domain pair mutants show limited fraying (Fig 5H).

Mutant filamin forms localize to the Z-disc We used the GFP tag in all the filamin mutants to test the domains required for Z-disc localization. Because the fluorescence from the endogenous filamin is low in the IFM Z-discs, we used the homozygote flies, which have both filamin copies tagged with GFP. Given that the myofibril phenotypes in the mutants are not present in all the sarcomeres, we analyzed normal-looking sarcomeres, ruptured sarcomeres, and enlarged Z-discs. In the control wild-type, filamin localizes to the Z-disc and diffusely to the cytoplasm (Fig 6A, red asterisks). The filaminclosed-GFP mutant also localizes to the Z-disc in normal-looking myofibrils (Fig 6B, red asterisks). In ruptured myofibrils, however, it accumulates at the ruptured Z-disc (Fig 6C and 6H). The filaminopen-GFP form mainly localizes to the enlarged Z-discs and the GFP signal from normal-looking Z-disc is weak but often detectable (Fig 6D and 6I). Interestingly, the signal from the Z-discs adjacent to the enlarged discs diminishes, suggesting that filamin molecules relocalize from normal Z-discs into the enlarged ones (Fig 6J). PPT PowerPoint slide

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TIFF original image Download: Fig 6. The filamin mutant forms localize to the Z-disc with different intensities. A-F) Confocal images of IFM from different homozygous filamin mutants. Actin filaments are shown in green, and GFP from the different filamin mutants in magenta. Red asterisks indicate selected Z-discs. Red arrowheads indicate ruptured discs. A) The filaminWT-GFP form localizes to the Z-discs (asterisks). B) The filaminclosed-GFP mutant form localizes to the Z-disc in normal-looking myofibrils (asterisks). C) When myofibrils get damaged, the filaminclosed-GFP mutant accumulates at the ruptured discs (arrowheads) and decreases in normal adjacent Z-discs. D) The filaminopen-GFP mutant accumulates mostly at the enlarged Z-discs and is barely detectable in the normal Z-discs. E) The filaminΔIg14-19-GFP mutant form localizes strongly in ruptured Z-discs and depletes filamin from adjacent discs. F) Removing the entire C-terminus part in filaminΔIg14-22-GFP mutant form does not prevent Z-disc localization in non-ruptured myofibrils. G) Boxplot of filamin GFP intensities in different filamin homozygous mutant backgrounds. H) GFP intensities in ruptured discs in various filamin homozygous mutant backgrounds. I) GFP intensities in enlarged discs in various filamin homozygous mutant backgrounds. J) GFP intensities in intact discs in filaminopen homozygous mutants, comparing those in proximity to enlarged discs versus those that are not. P-values were calculated using Welch’s two-sample t-test followed by a Bonferroni correction. In the Boxplots, the central box represents the 25–75th percentiles, and the median is indicated. The whiskers show the minimum and maximum values. https://doi.org/10.1371/journal.pgen.1011101.g006 The largest deletion mutant we have, filaminΔ14-22-GFP, has a clear Z-disc localization pattern in normal-looking myofibrils (Fig 6F), suggesting that the region responsible for Z-disc localization is in the N-terminal region anywhere between the CH-domains and Ig 13. The filamin mutant form lacking the entire MSR, filaminΔ14-19-GFP, accumulates at the ruptured Z-discs (Fig 6E). We then measured the intensity of GFP signal at individual Z-discs. Even though all the mutants localized to the Z-disc, all of them except for filaminΔ16-17-GFP and filaminΔ18-19-GFP had a diminished Z-disc signal compared to the control (Fig 6G). The accumulation of filamin in ruptured discs was similar among mutants that together span the entire C-terminal region (Fig 6H), suggesting filamin accumulation at ruptured Z-discs is mediated by the N-terminal half of filamin.

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