Carol C Gregorio
- Department Head, Cellular and Molecular Medicine
- Co-Director, Sarver Heart Center
- Director, Molecular Cardiovascular Research Program
- Professor, Cellular and Molecular Medicine
- Professor, Molecular and Cellular Biology
- Professor, BIO5 Institute
- Member of the Graduate Faculty
No activities entered.
No activities entered.
DissertationCMM 920 (Fall 2022)
Independent StudyCMM 599 (Summer I 2022)
DissertationCMM 920 (Spring 2022)
ResearchCMM 900 (Spring 2022)
DissertationCMM 920 (Fall 2021)
Independent StudyMCB 499 (Fall 2021)
ResearchCMM 900 (Fall 2021)
DissertationCMM 920 (Spring 2021)
Honors Independent StudyMCB 399H (Spring 2021)
ResearchCMM 900 (Spring 2021)
Directed ResearchMCB 792 (Fall 2020)
DissertationCMM 920 (Fall 2020)
Honors Independent StudyMCB 299H (Fall 2020)
ResearchCMM 900 (Fall 2020)
DissertationCMM 920 (Spring 2020)
DissertationCMM 920 (Fall 2019)
Introduction to ResearchMCB 795A (Fall 2019)
DissertationCMM 920 (Spring 2019)
ResearchCMM 900 (Spring 2019)
ResearchCMM 900 (Fall 2018)
Independent StudyMCB 399 (Spring 2018)
Independent StudyPSIO 499 (Spring 2018)
Independent StudyPSIO 399 (Fall 2017)
ResearchCMM 900 (Fall 2017)
ThesisCMM 910 (Fall 2017)
DissertationCMM 920 (Spring 2017)
Honors ThesisMCB 498H (Spring 2017)
ThesisCMM 910 (Spring 2017)
DissertationCMM 920 (Fall 2016)
Honors ThesisMCB 498H (Fall 2016)
Introduction to ResearchMCB 795A (Fall 2016)
ThesisCMM 910 (Fall 2016)
Directed RsrchMCB 392 (Spring 2016)
DissertationCMM 920 (Spring 2016)
Honors Independent StudyMCB 499H (Spring 2016)
Independent StudyMCB 399 (Spring 2016)
- Gregorio, C., Bliss, K. T., Chu, M., Jones-Weinert, C. M., & Gregorio, C. C. (2013). Investigating lasp-2 in cell adhesion: new binding partners and roles in motility. Molecular biology of the cell, 24(7).More infoFocal adhesions are intricate protein complexes that facilitate cell attachment, migration, and cellular communication. Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly identified component of these complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2: the integral focal adhesion proteins vinculin and paxillin. Of interest, the interaction of lasp-2 with its binding partners vinculin and paxillin is significantly reduced in the presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other; however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1. This suggests that the interplay between lasp-2 and lasp-1 could be an adhesion regulatory mechanism. Lasp-2's potential role in metastasis is revealed, as overexpression of lasp-2 in either SW620 or PC-3B1 cells-metastatic cancer cell lines-increases cell migration but impedes cell invasion, suggesting that the enhanced interaction of vinculin and paxillin may functionally destabilize focal adhesion composition. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.
- Gregorio, C., Nworu, C. U., Krieg, P. A., & Gregorio, C. C. (2013). Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging. Methods (San Diego, Calif.).More infoMutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (⩽ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy.
- Gregorio, C., Zarnescu, D. C., & Gregorio, C. C. (2013). Fragile hearts: New insights into translational control in cardiac muscle. Trends in cardiovascular medicine, 23(8).More infoCurrent investigations focused on RNA-binding proteins in striated muscle, which provide a scenario whereby muscle function and development are governed by the interplay of post-transcriptional RNA regulation, including transcript localization, splicing, stability, and translational control. New data have recently emerged, linking the RNA-binding protein FXR1 to the translation of key cytoskeletal components such as talin and desmoplakin in heart muscle. These findings, together with a plethora of recent reports implicating RNA-binding proteins and their RNA targets in both basic aspects of muscle development and differentiation as well as heart disease and muscular dystrophies, point to a critical role of RNA-based regulatory mechanisms in muscle biology. Here we focus on FXR1, the striated muscle-specific member of the Fragile X family of RNA-binding proteins and discuss its newly reported cytoskeletal targets as well as potential implications for heart disease.
- Gregorio, C., Conover, G. M., & Gregorio, C. C. (2011). The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments. Journal of cell science, 124(Pt 20).More infoDesmin intermediate filaments intimately surround myofibrils in vertebrate muscle forming a mesh-like filament network. Desmin attaches to sarcomeres through its high-affinity association with nebulin, a giant F-actin binding protein that co-extends along the length of actin thin filaments. Here, we further investigated the functional significance of the association of desmin and nebulin in cultured primary myocytes to address the hypothesis that this association is key in integrating myofibrils to the intermediate filament network. Surprisingly, we identified eight peptides along the length of desmin that are capable of binding to C-terminal modules 160-170 in nebulin. In this study, we identified a targeted mutation (K190A) in the desmin coil 1B region that results in its reduced binding with the nebulin C-terminal modules. Using immunofluorescence microscopy and quantitative analysis, we demonstrate that expression of the mutant desmin K190A in primary myocytes results in a significant reduction in assembled endogenous nebulin and desmin at the Z-disc. Non-uniform actin filaments were markedly prevalent in myocytes expressing GFP-tagged desmin K190A, suggesting that the near-crystalline organization of actin filaments in striated muscle depends on a stable interaction between desmin and nebulin. All together, these data are consistent with a model in which Z-disc-associated nebulin interacts with desmin through multiple sites to provide efficient stability to satisfy the dynamic contractile activity of myocytes.
- Gregorio, C., Pappas, C. T., Bliss, K. T., Zieseniss, A., & Gregorio, C. C. (2011). The Nebulin family: an actin support group. Trends in cell biology, 21(1).More infoNebulin, a giant, actin-binding protein, is the largest member of a family of proteins (including N-RAP, nebulette, lasp-1 and lasp-2) that are assembled in a variety of cytoskeletal structures, and expressed in different tissues. For decades, nebulin has been thought to act as a molecular ruler, specifying the precise length of actin filaments in skeletal muscle. However, emerging evidence suggests that nebulin should not be viewed as a ruler but as an actin filament stabilizer required for length maintenance. Nebulin has also been implicated recently in an array of regulatory functions independent of its role in actin filament length regulation. In this review, we discuss the current evolutionary, biochemical, and functional data for the nebulin family of proteins - a family whose members, both large and small, function as cytoskeletal scaffolds and stabilizers.
- Gregorio, C., Whitman, S. A., Cover, C., Yu, L., Nelson, D. L., Zarnescu, D. C., & Gregorio, C. C. (2011). Desmoplakin and talin2 are novel mRNA targets of fragile X-related protein-1 in cardiac muscle. Circulation research, 109(3).More infoThe proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied.
- Gregorio, C., Pappas, C. T., Krieg, P. A., & Gregorio, C. C. (2010). Nebulin regulates actin filament lengths by a stabilization mechanism. The Journal of cell biology, 189(5).More infoEfficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.
- Gregorio, C., Tsukada, T., Pappas, C. T., Moroz, N., Antin, P. B., Kostyukova, A. S., & Gregorio, C. C. (2010). Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle. Journal of cell science, 123(Pt 18).More infoRegulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.
- Gregorio, C., Conover, G. M., Henderson, S. N., & Gregorio, C. C. (2009). A myopathy-linked desmin mutation perturbs striated muscle actin filament architecture. Molecular biology of the cell, 20(3).More infoDesmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.
- Gregorio, C., Pappas, C. T., Bhattacharya, N., Cooper, J. A., & Gregorio, C. C. (2008). Nebulin interacts with CapZ and regulates thin filament architecture within the Z-disc. Molecular biology of the cell, 19(5).More infoThe barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160-164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.
- Gregorio, C., Zieseniss, A., Terasaki, A. G., & Gregorio, C. C. (2008). Lasp-2 expression, localization, and ligand interactions: a new Z-disc scaffolding protein. Cell motility and the cytoskeleton, 65(1).More infoThe nebulin family of actin-binding proteins plays an important role in actin filament dynamics in a variety of cells including striated muscle. We report here the identification of a new striated muscle Z-disc associated protein: lasp-2 (LIM and SH3 domain protein-2). Lasp-2 is the most recently identified member of the nebulin family. To evaluate the role of lasp-2 in striated muscle, lasp-2 gene expression and localization were studied in chick and mouse tissue, as well as in primary cultures of chick cardiac and skeletal myocytes. Lasp-2 mRNA was detected as early as chick embryonic stage 25 and lasp-2 protein was associated with developing premyofibril structures, Z-discs of mature myofibrils, focal adhesions, and intercalated discs of cultured cardiomyocytes. Expression of GFP-tagged lasp-2 deletion constructs showed that the C-terminal region of lasp-2 is important for its localization in striated muscle cells. Lasp-2 organizes actin filaments into bundles and interacts directly with the Z-disc protein alpha-actinin. These results are consistent with a function of lasp-2 as a scaffolding and actin filament organizing protein within striated muscle Z-discs.
- Gregorio, C., McElhinny, A. S., Schwach, C., Valichnac, M., Mount-Patrick, S., & Gregorio, C. C. (2005). Nebulin regulates the assembly and lengths of the thin filaments in striated muscle. The Journal of cell biology, 170(6).More infoIn many tissues, actin monomers polymerize into actin (thin) filaments of precise lengths. Although the exact mechanisms involved remain unresolved, it is proposed that "molecular rulers" dictate the lengths of the actin filaments. The giant nebulin molecule is a prime candidate for specifying thin filament lengths in striated muscle, but this idea has never been proven. To test this hypothesis, we used RNA interference technology in rat cardiac myocytes. Live cell imaging and triple staining revealed a dramatic elongation of the preexisting thin filaments from their pointed ends upon nebulin knockdown, demonstrating its role in length maintenance; the barbed ends were unaffected. When the thin filaments were depolymerized with latrunculin B, myocytes with decreased nebulin levels reassembled them to unrestricted lengths, demonstrating its importance in length specification. Finally, knockdown of nebulin in skeletal myotubes revealed its involvement in myofibrillogenesis. These data are consistent with nebulin functioning as a thin filament ruler and provide insight into mechanisms dictating macromolecular assembly.
- Gregorio, C., Ono, Y., Schwach, C., Antin, P. B., & Gregorio, C. C. (2005). Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation. Developmental biology, 282(2).More infoTropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.
- Gregorio, C., McElhinny, A. S., Perry, C. N., Witt, C. C., Labeit, S., & Gregorio, C. C. (2004). Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development. Journal of cell science, 117(Pt 15).More infoThe efficient functioning of striated muscle is dependent upon the structure of several cytoskeletal networks including myofibrils, microtubules, and intermediate filaments. However, little is known about how these networks function together during muscle differentiation and maintenance. In vitro studies suggest that members of the muscle-specific RING finger protein family (MURF-1, 2, and 3) act as cytoskeletal adaptors and signaling molecules by associating with myofibril components (including the giant protein, titin), microtubules and/or nuclear factors. We investigated the role of MURF-2, the least-characterized family member, in primary cultures of embryonic chick skeletal and cardiac myocytes. MURF-2 is detected as two species (approximately 55 kDa and approximately 60 kDa) in embryonic muscle, which are down-regulated in adult muscle. Although predominantly located diffusely in the cytoplasm, MURF-2 also colocalizes with a sub-group of microtubules and the M-line region of titin. Reducing MURF-2 levels in cardiac myocytes using antisense oligonucleotides perturbed the structure of stable microtubule populations, the intermediate filament proteins desmin and vimentin, and the sarcomeric M-line region. In contrast, other sarcomeric regions and dynamic microtubules remained unaffected. MURF-2 knock-down studies in skeletal myoblasts also delayed myoblast fusion and myofibrillogenesis. Furthermore, contractile activity was also affected. We speculate that some of the roles of MURF-2 are modulated via titin-based mechanisms.
- Gregorio, C., Miller, M. K., Granzier, H., Ehler, E., & Gregorio, C. C. (2004). The sensitive giant: the role of titin-based stretch sensing complexes in the heart. Trends in cell biology, 14(3).More infoEvery heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.
- Gregorio, C., Kazmierski, S. T., Antin, P. B., Witt, C. C., Huebner, N., McElhinny, A. S., Labeit, S., & Gregorio, C. C. (2003). The complete mouse nebulin gene sequence and the identification of cardiac nebulin. Journal of molecular biology, 328(4).More infoNebulin is a giant (M(r) 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulin's potential role as a molecular template is based on its structural and biochemical properties. Its central approximately 700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and approximately 80kDa of its C-terminal region integrates within the Z-line lattice. Here, we determined the exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segment. We identified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3 from its Z-line region). One novel exon shares high sequence homology to the 20 residue repeats of the tight-junction protein, ZO-1. RT-PCR analyses revealed that all 16 novel exons are expressed in mouse skeletal muscle. Surprisingly, we also amplified mRNA transcripts from mouse and human heart cDNA using primers designed along the entire length of nebulin. The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridization in fetal rat cardiomyocytes and in embryonic Xenopus laevis (frog) heart. On the protein level, antibodies specific for skeletal muscle nebulin's N and C-terminal regions stained isolated rat cardiac myofibrils at the pointed and barbed ends of thin filaments, respectively. These data indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal myofibrils. We propose that thin filament length regulation in cardiac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filament length differences in cardiac and skeletal muscle.
- Gregorio, C., McElhinny, A. S., Kazmierski, S. T., Labeit, S., & Gregorio, C. C. (2003). Nebulin: the nebulous, multifunctional giant of striated muscle. Trends in cardiovascular medicine, 13(5).More infoNebulin is a giant, modular sarcomeric protein and although it was discovered over 2 decades ago, it remains one of the most nebulous components of striated muscle. Previously, several groups identified nebulin as the prime candidate molecule for functioning as a "ruler" to specify the precise lengths of the actin (thin) filaments in skeletal muscle, yet this proposal has never been proven. This article reviews the evidence implicating nebulin as a thin filament ruler, including the most recent studies highlighting its potentially extensive isoform diversity and exciting reports revealing its expression in cardiac tissue. Also examined are novel findings indicating that nebulin is actually a multifunctional filament system, perhaps playing roles in signal transduction, contractile regulation, and myofibril force generation; these ideas are especially intriguing given the growing number of mutations in this giant molecule that are associated with human myopathies.
- Gregorio, C., Mudry, R. E., Perry, C. N., Richards, M., Fowler, V. M., & Gregorio, C. C. (2003). The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes. The Journal of cell biology, 162(6).More infoActin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.
- Gregorio, C., Clark, K. A., McElhinny, A. S., Beckerle, M. C., & Gregorio, C. C. (2002). Striated muscle cytoarchitecture: an intricate web of form and function. Annual review of cell and developmental biology, 18.More infoStriated muscle is an intricate, efficient, and precise machine that contains complex interconnected cytoskeletal networks critical for its contractile activity. The individual units of the sarcomere, the basic contractile unit of myofibrils, include the thin, thick, titin, and nebulin filaments. These filament systems have been investigated intensely for some time, but the details of their functions, as well as how they are connected to other cytoskeletal elements, are just beginning to be elucidated. These investigations have advanced significantly in recent years through the identification of novel sarcomeric and sarcomeric-associated proteins and their subsequent functional analyses in model systems. Mutations in these cytoskeletal components account for a large percentage of human myopathies, and thus insight into the normal functions of these proteins has provided a much needed mechanistic understanding of these disorders. In this review, we highlight the components of striated muscle cytoarchitecture with respect to their interactions, dynamics, links to signaling pathways, and functions. The exciting conclusion is that the striated muscle cytoskeleton, an exquisitely tuned, dynamic molecular machine, is capable of responding to subtle changes in cellular physiology.
- Gregorio, C., McElhinny, A. S., Kakinuma, K., Sorimachi, H., Labeit, S., & Gregorio, C. C. (2002). Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1. The Journal of cell biology, 157(1).More infoThe COOH-terminal A168-170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted. Disruption of titin's M-line region also resulted in a perturbation of thick filament components, but, surprisingly, not of the NH2-terminal or I-band regions of titin, the Z-lines, or the thin filaments. This specific phenotype also was caused by the expression of titin A168-170. These data suggest that the interaction of titin with MURF-1 is important for the stability of the sarcomeric M-line region.MURF-1 also binds to ubiquitin-conjugating enzyme-9 and isopeptidase T-3, enzymes involved in small ubiquitin-related modifier-mediated nuclear import, and with glucocorticoid modulatory element binding protein-1 (GMEB-1), a transcriptional regulator. Consistent with our in vitro binding data implicating MURF-1 with nuclear functions, endogenous MURF-1 also was detected in the nuclei of some myocytes. The dual interactions of MURF-1 with titin and GMEB-1 may link myofibril signaling pathways (perhaps including titin's kinase domain) with muscle gene expression.
- Gregorio, C., McElhinny, A. S., Kolmerer, B., Fowler, V. M., Labeit, S., & Gregorio, C. C. (2001). The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments. The Journal of biological chemistry, 276(1).More infoStrict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.
- Gregorio, C., Rudy, D. E., Yatskievych, T. A., Antin, P. B., & Gregorio, C. C. (2001). Assembly of thick, thin, and titin filaments in chick precardiac explants. Developmental dynamics : an official publication of the American Association of Anatomists, 221(1).More infoDe novo cardiac myofibril assembly has been difficult to study due to the lack of available cell culture models that clearly and accurately reflect heart muscle development in vivo. However, within precardiac chick embryo explants, premyocardial cells differentiate and commence beating in a temporal pattern that corresponds closely with myocyte differentiation in the embryo. Immunofluorescence staining of explants followed by confocal microscopy revealed that distinct stages of cardiac myofibril assembly, ranging from the earliest detection of sarcomeric proteins to the late appearance of mature myofibrils, were consistently recognized in precardiac cultures. Assembly events involved in the early formation of sarcomeres were clearly visualized and accurately reflected observations described by others during chick heart muscle development. Specifically, the early colocalization of alpha-actinin and titin dots was observed near the cell periphery representing I-Z-I-like complex formation. Myosin-containing thick filaments assembled independently of actin-containing thin filaments and appeared centered within sarcomeres when titin was also linearly aligned at or near cell borders. An N-terminal epitope of titin was detected earlier than a C-terminal epitope; however, both epitopes were observed to alternate near the cell periphery concomitant with the earliest formation of myofibrils. Although vascular actin was detected within cells during early assembly stages, cardiac actin predominated as the major actin isoform in mature thin filaments. Well-aligned thin filaments were also observed in the absence of organized staining for tropomodulin at thin filament pointed ends, suggesting that tropomodulin is not required to define thin filament lengths. Based on these findings, we conclude that the use of the avian precardiac explant system accurately allows for direct investigation of the mechanisms regulating de novo cardiac myofibrillogenesis.
- Gregorio, C., McElhinny, A. S., Labeit, S., & Gregorio, C. C. (2000). Probing the functional roles of titin ligands in cardiac myofibril assembly and maintenance. Advances in experimental medicine and biology, 481.More infoSarcomeres of cardiac muscle are comprised of numerous proteins organized in an elegantly precise order. The exact mechanism of how these proteins are assembled into myofibrils during heart development is not yet understood, although existing in vitro and in vivo model systems have provided great insight into this complex process. It has been proposed by several groups that the giant elastic protein titin acts as a "molecular template" to orchestrate sarcomeric organization during myofibrillogenesis. Titin's highly modular structure, composed of both repeating and unique domains that interact with a wide spectrum of contractile and regulatory ligands, supports this hypothesis. Recent functional studies have provided clues to the physiological significance of the interaction of titin with several titin-binding proteins in the context of live cardiac cells. Improved models of cardiac myofibril assembly, along with the application of powerful functional studies in live cells, as well as the characterization of additional titin ligands, is likely to reveal surprising new functions for the titin third filament system.
- Gregorio, C., & Gregorio, C. C. (1997). Models of thin filament assembly in cardiac and skeletal muscle. Cell structure and function, 22(1).More infoThe assembly and functional characteristics of many contractile proteins are different in skeletal and cardiac muscle. Two models for thin filament assembly are consistent with observations from recent studies focused on determining the functional significance of actin filament capping in primary cultures of embryonic chick myogenic cells and cardiac myocytes. Future experiments will test the validity of the proposed models for in vivo embryonic development.
- Pappas, C. T., Farman, G. P., Mayfield, R. M., & Gregorio, C. C. (2017, December). Leiomodin-2 regulates thin filament assembly and is necessary for proper contractile force production in the hearts of adult mice. American Society of Cell Biology/European Molecular Biology Organization Annual Meeting.