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Clinical correlates for WHO II and III CP tumors and PTPR. Figure S1. Comparison between the expression of NHERF1, moesin and NF2 in normal CP and CP papilloma (CPP). IHC with indicated antibodies of serial sections from a resection specimen (1 year-old female) containing both normal CP (right) and CP papilloma (left). Note higher expression of all 3 proteins in normal CP than in papilloma and cytoplasmic NHERF1 expression in normal CP but not in CP papilloma. Figure S2. The staining profiles of CP papilloma with oncocytic-like change and normal CP are similar. IHC with indicated antibodies of serial sections (×20 original magnification) from a resection specimen (22 year-old male) containing CP papilloma with (left) or without (upper right) oncocytic-like change. Figure S3. NHERF1 IHC distinguishes compact areas in CP papilloma (CPP) and carcinoma (CPC). Compact areas may show ill-defined papillary architecture in cases of CPP (6 year-old female) and CPC (1 year-old male). NHERF1 preserves the characteristic IHC pattern in CPP and shows faint cytoplasmic and focal membranous staining in this CPC example with extensive necrosis. Also shown are NF2 membrane staining and mitoses (arrows) (insets, 40× original magnification). Figure S4. NHERF1 IHC highlights normal CP in a resection speciment of CP papilloma. Serial sections (×10 original magnification) from a resection specimen (5 month-old female) containing both normal CP (right) and CP papilloma (left) show clear demarcation by NHERF1 IHC. Note also continuity of normal CP, at the tip, and CP papilloma, at the base, of the same major papillary stem. Figure S5. NF2 IHC in compact and papillary areas from CP papilloma (CPP) and PTPR, as compared to ependymoma. IHC with NF2 antibody shows distinct basolateral and apical plasma membrane staining in compact and papillary areas of CPP (same case as in Fig. S3). NF2 is cytoplasmic in compact areas from PTPR and membranous in papillary areas. Strong plasma membrane moesin staining is also noted in papillary areas in PTPR. NF2 is cytoplasmic in ependymoma. Very faint NF2 membranous staining of polarized structures may be seen in PTPR (rosette lumens in this example) or ependymoma (microlumens and rings). In the example of ependymoma, the same area in a serial section stained with NHERF1 antibody shows rings and microlumens. Figure S6. Example of area with rosettes in ependymoma (WHO grade II). H&E and NHERF1 IHC of serial sections from a resection specimen (5 month-old female) showing labeling of rosette lumens (arrows) and of microlumens. (PDF 1266 kb)

Background The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin–amphiphysin–Rvs (BAR)–pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair. Methods We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice. Results Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin. Conclusions Overall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients.