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Competent human DNA mismatch repair (MMR) corrects DNA polymerase mistakes made during cell replication to maintain complete DNA fidelity in daughter cells; faulty DNA MMR occurs in the setting of inflammation and neoplasia, creating base substitutions (e.g. point mutations) and frameshift mutations at DNA microsatellite sequences in progeny cells. Frameshift mutations at DNA microsatellite sequences are a detected biomarker termed microsatellite instability (MSI) for human disease, as this marker can prognosticate and determine therapeutic approaches for patients with cancer. There are two types of MSI: MSI-High (MSI-H), defined by frameshifts at mono- and di-nucleotide microsatellite sequences, and elevated microsatellite alterations at selected tetranucleotide repeats or EMAST, defined by frameshifts in di- and tetranucleotide microsatellite sequences but not mononucleotide sequences. Patients with colorectal cancers (CRCs) manifesting MSI-H demonstrate improved survival over patients without an MSI-H tumor, driven by the generation of immunogenic neoantigens caused by novel truncated proteins from genes whose sequences contain coding microsatellites; these patients’ tumors contain hundreds of somatic mutations, and show responsiveness to treatment with immune checkpoint inhibitors. Patients with CRCs manifesting EMAST demonstrate poor survival over patients without an EMAST tumor, and may be driven by a more dominant defect in double strand break repair attributed to the MMR protein MSH3 over its frameshift correcting function; these patients’ tumors often have a component of inflammation (and are also termed inflammation-associated microsatellite alterations) and show less somatic mutations and lack coding mononucleotide frameshift mutations that seem to generate the neoantigens seen in the majority of MSI-H tumors. Overall, both types of MSI are biomarkers that can prognosticate patients with CRC, can be tested for simultaneously in marker panels, and informs the approach to specific therapy including immunotherapy for their cancers.

During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues.

Background. Oxidative DNA damage and inflammatory cytokines cause loss of function of the MMR protein MSH3. MSH3-MSH2 (MutSβ) function is required for correction of postreplicative polymerase errors in the form of frameshifts at tetranucleotide microsatellite sequences to prevent elevated microsatellite alterations at selected tetranucleotides (EMAST). EMAST is increasingly being used as a biomarker for MSH3 deficiency in subtyping colorectal cancers due to its association with metastasis and poor patient outcome. Here, we sought to understand frameshift mutational behavior of tetranucleotide sequences in the absence of MMR. Methods. We generated EGFP-based mutation reporter model systems containing the D9S242 tetranucleotide microsatellite at its native length of (AAAG)18 and at modified lengths of (AAAG)15 and (AAAG)12 to represent other endogenous microsatellite lengths. Plasmid vectors were constructed for each length that placed EGFP +1 bp or -1 bp out-of-frame for protein translation, enabling us to measure deletion and insertion frameshifts. These constructs were stably-integrated into MMR-deficient HCT116 cells, and monoclonal cell lines containing the constructs were isolated to measure mutation rates and spectra. Deletion and insertion frameshift mutations restored the EGFP reading frame to become detectable by flow cytometry. Genomic DNA from mutated cells were isolated and sequenced for insertion/deletion frameshift mutational biases. Results. Combined insertion/deletion frameshift mutation rates at D9S242 locus ranged from 31.6 X 10-4 to 71.1 X 10-4 mutations/cell/generation and was strongly correlated with longer length of tetranucleotide microsatellites (r2 = 0.986, P = 0.0375). We observed slightly higher deletion mutation rates over insertion rates at longer microsatellites, but equivalent insertion and deletion rates at shorter microsatellites. However, accumulation of more deletion frameshifts over time contributed to a distinct mutational bias for deletions for each length, likely due to continual frameshift mutation at insertions. About 79% of all observed frameshifts (insertion or deletion) were one- repeat (e.g., four nucleotides), 16% two-repeat, and 5% three or more repeat mutations that were consistent with a slipped strand mispairing mutation model. Conclusions. Our results suggest that intact MMR recognizes and repairs shorter length tetranucleotide frameshifts more efficiently than larger ones, and preferentially corrects loops on the template strand (preventing deletions) over loops on the newly-synthesized strand (preventing insertions) in human cells. MMR deficiency thus manifests in human tissue with a deletion mutation bias at tetranucleotide sequences (EMAST). Longer tetranucleotide microsatellites have increased propensities for larger DNA slippage errors. Citation Format: Maide O. Raeker, Jovan Pierre-Charles, John M. Carethers. Insertion and deletion frameshift rates and mutational spectra of tetranucleotide microsatellites in DNA mismatch repair-deficient human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3368.

Obscurin is a giant structural and signaling protein that participates in the assembly and structural integrity of striated myofibrils. Previous work has examined the physical interactions between obscurin and other cytoskeletal elements but its in vivo role in cell signaling, including the functions of its RhoGTPase Exchange Factor (RhoGEF) domain have not been characterized. In this study, morpholino antisense oligonucleotides were used to create an in-frame deletion of the active site of the obscurin A RhoGEF domain in order to examine its functions in zebrafish development. Cardiac myocytes in the morphant embryos lacked the intercalated disks that were present in controls by 72 and, in the more severely affected embryos, the contractile filaments were not organized into mature sarcomeres. Neural abnormalities included delay or loss of retinal lamination. Rescue of the phenotype with co-injection of mini-obscurin A expression constructs demonstrated that the observed effects were due to the loss of small GTPase activation by obscurin A. The immature phenotype of the cardiac myocytes and the retinal neuroblasts observed in the morphant embryos suggests that obscurin A-mediated small GTPase signaling promotes tissue-specific cellular differentiation. This is the first demonstration of the importance of the obscurin A-mediated RhoGEF signaling in vertebrate organogenesis and highlights the central role of obscurin A in striated muscle and neural development.

Obscurin and obscurin-associated kinase are two products of the obscurin transcriptional unit that encodes a recently identified giant muscle-specific protein obscurin. In this study, we characterized the developmental expression and cellular localization of obscurin and obscurin-associated kinase in cardiac muscle cells. We cloned murine obscurin-associated kinase and found that it is abundantly expressed in the heart as two isotypes encoded by 2.2 and 4.9 kb sequences. The 2.2 kb isotype of the kinase was more prominently expressed than the 4.9 kb isotype. Both obscurin and the kinase-like domains were progressively upregulated since the early stages of cardiac development. Obscurin-associated kinase was expressed at higher levels than obscurin at early stages of cardiomyogenesis. Increasing intensity of obscurin expression in the developing heart positively correlated with progressive cell differentiation and was higher in the ventricles compared to the atria. These data were supported by the results of experiments with primary cardiac cell cultures. Obscurin localization changed from a weakly immunopositive diffuse pattern in poorly differentiated cells to an intensely immunolabeled cross-striated distribution at the level of mid-A-bands and Z-disks during the assembly of the myofibrillar contractile apparatus. In dividing myocytes, unlike the interphase cells, obscurin translocated from disassembling myofibrils into a diffuse granulated pattern segregated separately from alpha-actinin-immunopositive aggregates. Obscurin-associated kinase was localized mainly to cell nuclei with increasing incorporation into the Z-disks during differentiation. Our results suggest that these two novel proteins are involved in the progression of cardiac myogenesis during the transition to advanced stages of heart development.

Recurrent 2q13 deletion syndrome is associated with incompletely penetrant severe cardiac defects and craniofacial anomalies. We used an atypical, overlapping 1.34 Mb 2q13 deletion in a patient with pathogenically similar congenital heart defects (CHD) to narrow the putative critical region for CHD to 474 kb containing six genes. To determine which of these genes is responsible for severe cardiac and craniofacial defects noted in the patients with the deletions, we used zebrafish morpholino knockdown to test the function of each orthologue during zebrafish development. Morpholino-antisense-mediated depletion of fibulin-7B, a zebrafish orthologue of fibulin-7 (FBLN7), resulted in cardiac hypoplasia, deficient craniofacial cartilage deposition and impaired branchial arch development. TMEM87B depletion likewise resulted in cardiac hypoplasia but with preserved branchial arch development. Depletion of both fibulin-7B and TMEM87B resulted in more severe defects of cardiac development, suggesting that their concurrent loss may enhance the risk of a severe cardiac defect. We postulate that heterozygous loss of FBLN7 and TMEM87B account for some of the clinical features, including cardiac defects and craniofacial abnormalities associated with 2q13 deletion syndrome.

Striated muscle has a highly ordered structure in which specialized domains of the cell membrane involved in force transmission (costameres) and excitation-contraction coupling (T tubules) as well as the internal membranes of the sarcoplasmic reticulum are organized over specific regions of the sarcomere. Optimal muscle function is dependent on this high level of organization but how it established and maintained is not well understood. Due to its ex utero development and transparency, the zebrafish embryo is an excellent vertebrate model for the study of dynamic relationships both within and between cells during development. Transgenic models have allowed the delineation of cellular migration and complex morphogenic rearrangements during the differentiation of skeletal myocytes and the assembly and organization of new myofibrils. Molecular targeting of genes and transcripts has allowed the identification of key requirements for myofibril assembly and organization. With the recent advances in gene editing approaches, the zebrafish will become an increasingly important model for the study of human myopathies and muscular dystrophies. Its high fecundity and small size make it well suited to high-throughput screenings to identify novel pharmacologic and molecular therapies for the treatment of a broad range of neuromuscular conditions. In this review, we examine the lessons learned from the zebrafish model regarding the complex interactions between the sarcomere and the sarcolemma that pattern the developing myocyte and discuss the potential for zebrafish as a model system to examine the pathophysiology of, and identify new treatments for, human myopathies and muscular dystrophies.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.

Myosin light chain kinases (MLCK) are a family of signaling proteins that are required for cytoskeletal remodeling in myocytes. Recently, two novel MLCK proteins, SPEG and obscurin-MLCK, were identified with the unique feature of two tandemly-arranged MLCK domains. In this study, the evolutionary origins of this MLCK subfamily were traced to a probable orthologue of obscurin-MLCK in Drosophila melanogaster, Drosophila Unc-89, and the MLCK kinase domains of zebrafish SPEG, zebrafish obscurin-MLCK, and human SPEG were characterized. Phylogenetic analysis of the MLCK domains indicates that the carboxy terminal kinase domains of obscurin-MLCK, SPEG and Unc-89 are more closely related to each other than to the amino terminal kinase domains or to other MLCKs, supporting the assertion that obscurin-MLCK is the vertebrate orthologue of Caenorhabditis elegans Unc-89, a giant multidomain protein that is required for normal myofibril assembly. The apparent lack of an invertebrate orthologue of SPEG and the conserved exon structure of the kinase domains between SPEG and obscurin-MLCK suggests that SPEG arose from obscurin-MLCK by a gene duplication event. The length of the primary amino acid sequence between the immunoglobulin (Ig) domains associated with the MLCK motifs is conserved in obscurin-MLCK, SPEG and C. elegans Unc-89, suggesting that these putative protein interaction domains may target the kinases to highly conserved intracellular sites. The conserved arrangement of the tandem MLCK domains and their relatively restricted expression in striated muscle indicates that further characterization of this novel MLCK subfamily may yield important insights into cardiac and skeletal muscle physiology.

Obscurin and obscurin myosin light chain kinase (MLCK) are two recently identified muscle proteins encoded by the same gene cluster. The production of obscurin, which contains a Rho-guanine exchange factor (GEF)-like sequence, and obscurin-MLCK by this cluster suggests that these novel genes may be involved in signal transduction cascades that control adaptive and compensatory responses of the heart. The goal of the present study was to investigate the transcriptional response of the obscurin gene cluster to the initiation of myocardial hypertrophy induced in mice by aortic constriction. The transcriptional activity of the obscurin genes was examined using reverse-transcriptase primed quantitative PCR. We found that the transcripts encoding the obscurin Rho-GEF and the obscurin-MLCK internal serine–threonine kinase II (SK II) domains were significantly upregulated following aortic constriction. The expression of Rho-GEF-containing transcripts at different stages of the hypertrophic growth exceeded the control levels by 2- to 6-fold. Following the induction of hypertrophy, the quantity of the SK II-encoding transcripts increased 10-fold by 24 h and 16-fold by 48 h, then decreased by day 7, and returned to the control level by day 56. The quantity of the carboxy terminal obscurin-MLCK transcripts encoding for SK I increased 2-fold by day 2 and returned to the control values at later stages. Immunolocalization of obscurin, which contains Rho-GEF domain, in cardiomyocytes during pharmacologically induced hypertrophic growth in vitro demonstrated that the expression was topographically associated with the growing myofibrils and with the sites of initiation and progression of myofibrillogenesis at the periphery of the sarcoplasm. This suggests that upregulation of obscurin synthesis is associated with the formation of additional amounts of contractile structures during cardiac hypertrophy. Thus, the obscurin gene cluster represents a new example of an operon that encodes differentially regulated structural and signaling proteins implicated in the control of assembly and adaptive remodeling of myofibrils during normal and hypertrophic growth.

Members of the Dbl family of guanine nucleotide exchange factors (GEFs) have important roles in the organization of actin-based cytoskeletal structures of a wide variety of cell types. Through the activation of members of the Rho family of GTP signaling molecules, these exchange factors elicit cytoskeletal alterations that allow cellular remodeling. As important regulators of RhoGTPase activity, members of this family are candidates for mediating the RhoGTPase activation and cytoskeletal changes that occur during cardiac development and during the myocardial response to hypertrophic stimuli. In this study, we characterize a novel human gene that is expressed in skeletal and cardiac muscle and has putative functional domains similar to those found in members of both the Dbl family of GEFs and the titin family of myosin light chain kinases (MLCK). The cDNA sequence of this gene, which has been designated Obscurin-myosin light chain kinase (Obscurin-MLCK), would be predicted to encode for at least 68 immunoglobulin domains, two fibronectin domains, one calcium/calmodulin binding domain, a RhoGTP exchange factor domain, and two serine-threonine kinase domains. The combination of the putative Rho GEF and two kinase domains has not been noted in any other members of the titin or Dbl families. Alternative splicing allows the generation of a number of unique Obscurin-MLCK isoforms that contain various combinations of the functional domains. One group of isoforms is comparable to Unc-89, a Caenorhabditis elegans sarcomere-associated protein, in that they contain a putative RhoGEF domain and multiple immunoglobulin repeats. Other isoforms more closely resemble MLCK, containing one or both of the putative carboxy-terminal serine-threonine kinase domains. The modular nature of the Obscurin-MLCK isoforms indicates that it may have an array of functions important to cardiac and skeletal muscle physiology.

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell–cell and cell–matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell–matrix, cell–cell, and intracellular cytoskeletal connections.

During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well organized connective tissue attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that apical clustering of integrins was associated with changes in cell morphology which included myoblast elongation along the body axis. Through the analysis of zebrafish embryos depleted of obscurin A that have impaired formation of the myotendinous junctions, we determined that cell elongation and, as a result, new myofibril formation was delayed in regions that lacked integrin clustering and fibronectin matrix organization. In addition, it was noted that striated myofibrils first form at the cell periphery and are associated with the early patterning of the lateral sarcolemma. These specialized membrane domains will ultimately form mature lateral connections between the myofibril and the extracellular matrix at the costameres. We have also determined that the giant cytoskeletal and sarcomeric protein obscurin has an important role in the organization and maturation of the MTJ and in the maturation of the costamere. As myocyte-extracellular matrix connections have critical roles in force transduction and tension-mediated signaling defining the processes that promote and sustain these connections will have important implications for the understanding the pathogenesis of, and developing new treatment strategies for, a range of myopathies and muscular dystrophies.