1985 — 1987 |
Wang, Kuan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biochemistry of New Myofibrillar Proteins @ University of Texas Austin
We propose to continue our studies on the molecular properties and the sarcomeric organization of two extremely large, major myofibrillar proteins\-- titin and nebulin -- which are found in the skeletal and cardiac muscles of a wide range of vertebrate and invertebrate species. Our studies in the past years have indicated that titin and nebulin may be components of an elastic, continuous myofilament (the "third" filament) -- distinct from this and thick filaments -- within the sarcomere. In the next grant period, we propose to (a) investigate further the molecular properties of titin and nebulin emphasizing the purification of native proteins, the study of domain organization, the study of immunological properties, the visualization of morphology and the investigation of their interactions with other myofibrillar proteins; (b) continue and extend our antibody localization studies to include the use of monoclonal antibodies and electron microscopic techniques, emphasizing the detection and labeling of ultrathin filaments in the sarcomere; (c) characterize the phosphorylation and calcium binding properties of titin and nebulin, emphasizing the possible involvement of these proteins in regulatory mechanisms; and (d) perform a survey of the distribution and organization of titin and nebulin in nature, emphasizing their presence in insect flight muscle and in nonmuscle cells. We hope that the biochemical and structural studies proposed here will lead to better understanding of the structure and function of contractile machinery, the mechanical properties of striated muscle and the mechanism of muscle contraction and relaxation.
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1 |
1985 — 1988 |
Wang, Kuan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Cytoskeletal Proteins in Platelet Physiology @ University of Texas Austin
Platelets, the smallest components of peripheral blood, play an essential role in hemostasis. When platelets are exposed to damaged vessels, they adhere to the exposed subendothelial collagen, change shape and release substances which are responsible for the subsequent aggregation of platelets, ultimately leading to the formation of a thrombus. The platelet contractile proteins and cytoskeleton have been proposed as providing the motile force responsible for both the morphological changes and the active secretion of granule contents. The molecular basis of the motile mechanism remain obscrue. This proposal, representing an extension of an on-going research program, addresses the question of how two major contractile proteins, filamin and P235 (a major platelet protein recently characterized in this laboratory) are involved in the transient and dynamic cytoplasmic cytoskeleton of human platelets. Specifically, we propose (1) to carry out a detailed biophysical characterization of filamin-actin and P235-actin interactions to evaluate the hypothesis that filamin is involved in the organization of actin-containing microfilaments, and that P235 is involved in maintaining the nonfilamentous or profilamentous state of actin; (2) to study the organization and protein association in intact platelet and in the isolated cytoskeleton by antibody localization techniques and by chemical crosslinking techniques.
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1 |
1988 |
Wang, Kuan |
S15Activity Code Description: Undocumented code - click on the grant title for more information. |
Small Instrumentation Program @ University of Texas Austin
biomedical equipment resource;
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1 |
1990 — 1993 |
Wang, Kuan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Proteins &Architecture of An Elastic Sarcomere Matrix @ University of Texas Austin
The long range goal of this proposal is to understand the structural and physiological roles of sarcomere-associated cytoskeletons in the striated muscle cells. Recent studies of two giant structural proteins, titin, and nebulin, suggest that each protein constitutes a set of molecular filaments, forming an elastic matrix in the sarcomere. The sarcomere matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability and the assembly of nascent sarcomeres in developing muscle cells. In the proposed research, we address several questions of central importance and examine them systematically and critically with an integrated experimental approach: (a) What is the conformational basis of titin elasticity? We will test the hypothesis that specific and reversible conformational transition between folded and unfolded states of titin polypeptides underlies its capacity as a molecular spring to generate elasticity. The molecular structure and its conformational transition will be determined at the level of cDNA sequence, amino acid sequence, protein folding, domain organization, contour length, flexibility and extensibility under a variety of experimental conditions. (b) Do titin and nebulin serve as templates or scaffolds for myosin and actin filaments, respectively? We will search for specific interfilament protein interactions and test their effects on actomyosin interaction. Additionally, their potential involvement in the assembly and length regulation of myosin and actin filaments will be examined at the level of self-assembly, nucleated assembly and reconstitution of selectively extracted sarcomeres? (c) How are titin and nebulin organized in sarcomere? We will evaluate a four-filament sarcomere model by high resolution electron microscopy combined with immuno-labeling. Additionally, a systematic search for binding proteins at the Z-and M-lines will be made t elucidate their anchorage in the sarcomere. (d) How does the sarcomere matrix respond to stress? We will evaluate and refine our working hypothesis that the segmental extensibility of titin underlies its capacity as a dual-range molecular spring. A battery of monoclonal antibodies to non-repetitive epitopes will be used to track dynamic translocations and filament strain in sarcomeres of various lengths. Experimental conditions will be designed to facilitate correlation between molecular conformation, sarcomere architecture and muscle mechanics of skeletal and cardiac muscles.
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1 |
1994 |
Wang, Kuan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Proteins and Architecture of An Elastic Sarcomere Matrix @ University of Texas Austin
The long range goal of this proposal is to understand the structural and physiological roles of sarcomere-associated cytoskeletons in the striated muscle cells. Recent studies of two giant structural proteins, titin, and nebulin, suggest that each protein constitutes a set of molecular filaments, forming an elastic matrix in the sarcomere. The sarcomere matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability and the assembly of nascent sarcomeres in developing muscle cells. In the proposed research, we address several questions of central importance and examine them systematically and critically with an integrated experimental approach: (a) What is the conformational basis of titin elasticity? We will test the hypothesis that specific and reversible conformational transition between folded and unfolded states of titin polypeptides underlies its capacity as a molecular spring to generate elasticity. The molecular structure and its conformational transition will be determined at the level of cDNA sequence, amino acid sequence, protein folding, domain organization, contour length, flexibility and extensibility under a variety of experimental conditions. (b) Do titin and nebulin serve as templates or scaffolds for myosin and actin filaments, respectively? We will search for specific interfilament protein interactions and test their effects on actomyosin interaction. Additionally, their potential involvement in the assembly and length regulation of myosin and actin filaments will be examined at the level of self-assembly, nucleated assembly and reconstitution of selectively extracted sarcomeres? (c) How are titin and nebulin organized in sarcomere? We will evaluate a four-filament sarcomere model by high resolution electron microscopy combined with immuno-labeling. Additionally, a systematic search for binding proteins at the Z-and M-lines will be made t elucidate their anchorage in the sarcomere. (d) How does the sarcomere matrix respond to stress? We will evaluate and refine our working hypothesis that the segmental extensibility of titin underlies its capacity as a dual-range molecular spring. A battery of monoclonal antibodies to non-repetitive epitopes will be used to track dynamic translocations and filament strain in sarcomeres of various lengths. Experimental conditions will be designed to facilitate correlation between molecular conformation, sarcomere architecture and muscle mechanics of skeletal and cardiac muscles.
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1 |
1995 — 1997 |
Wang, Kuan |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Structure and Function of Skeletal Muscle Nebulin @ University of Texas Austin
The long term goal of our proposed research is to understand the molecular structure of nebulin and to define how nebulin contributes to the mechanical properties and developmental programs of skeletal muscle cells. Nebulin is a giant actin binding protein that comprises nearly 2% of myofibrillar proteins. This modular protein has been proposed to act as a protein ruler to regulate the length of thin filaments, as a calcium- calmodulin-mediated regulatory protein of actomyosin interaction, and as a template for the assembly of the thin filament lattice during development. We propose to evaluate these hypotheses critically using a combination of biochemical, biophysical, structural and cellular approaches. The specific aims are: (l) to correlate the size of nebulin isoforms and the length of actin filaments in a wide range of skeletal muscles. Scanning transmission electron microscopy and mass spectrometry will be used to help determine the size and contour length of nebulin isoforms of mature and developing skeletal muscles of rabbit, mouse, and chicken. lmmunolocalization of actin and tropomyosin will be used to help define the length of actin filaments. (2) to prepare nebulin-containing thin filaments and study the arrangement of nebulin on the thin filaments by high resolution cryoelectron microscopy. (3) to investigate the mechanisms of nebulin-actin interaction and nebulin's inhibition of actomyosin interaction, its reversal by calcium and calmodulin and its potential regulation by phosphorylation and phospholipids. Cosedimentation, solid phase binding, ATPase assays, in vitro motility assays, and fluorescence spectroscopy will be used to reveal molecular interactions. (4) to define molecular interfaces of nebulin with actin, myosin, and calmodulin. Chemical crosslinking, protein sequencing and mutant nebulin fragments will he used to identify important amino acids at the interface. (5) to complete the coding sequence of human skeletal muscle nebulin, to define the mechanisms with which nebulin isoforms are generated and to catalog functional domains. (6) to elucidate the role of nebulin in myofibrillogenesis in developing mouse skeletal muscle in culture. The synthesis and assembly of nebulin and its transcripts in normal cells and in cells that overexpress nebulin fragments will be monitored, and thin filament length will be determined by immunoelectron microscopy. The presence of nebulin in the contractile machinery of skeletal muscles poses new challenges and elicits renewed interest in the understanding of thin filaments. The proposed research would define the physiological function of nebulin and the molecular basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.
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1 |
1995 — 1997 |
Wang, Kuan |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of a Confocal Microscope Imaging System @ University of Texas At Austin
The specific objectives of this proposal are to: 1) Determine the functional domains of the subunits of wheat germ eIF-(iso)4F and eIF-4F. The subunits of eIF-(iso)4F have been expressed in E. coli and recombined to form an enzymatically active complex. Mutations will be made in the subunits of eIF-(iso)4F and eIF-4F and tested in an in vitro wheat germ translation system and several in vitro assays that measure partial reactions, i.e., ATP hydrolysis, RNA unwinding, cross-linking to the m7G cap and binding of mRNA to 40S ribosomal subunits. 2) Complete the partial cDNA sequence of wheat eIF-4B and express this protein in E. coli. The missing portion ( 100-200 nucleotides) of the cDNA for eIF-4B will be obtained by RACE-PCR, inverse PCR or genomic sequencing. The cDNA will be expressed in E. coli and the enzymatic activity determined in the assays described above (Specific Objective 1). 3) Complete the cDNA sequence for the subunits of Arabidopsis eIF-4F, p28 of eIF-(iso)4F and eIF-4B and express these proteins in E. coli. We will continue to screen using antibodies to wheat factors and wheat cDNA sequences. Different regions of the wheat cDNAs will be used to prepare probes for screening. The cDNAs for the Arabidopsis factors will be expressed in E. coli. The activity of the expressed Arabidopsis factors will be measured in the assays described above. The completion of this goal will set the stage for future work in vivo exploring the control of expression of the initiation factors and regulation of protein synthesis in plants.
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1 |
1996 |
Wang, Kuan |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Nebulin as a Molecular Ruler of Thin Filaments @ University of Texas Austin
DESCRIPTION The long-term goal of the proposed research is to understand the physiological and developmental roles of nebulin isoforms in vertebrate skeletal muscles, which have been identified by variability in the molecular mass (600-900 kD). This giant modular protein has been proposed to act as a protein ruler to regulate the length of thin filaments, as a calcium-calmodulin mediated regulatory protein of actomyosin interaction, and as a template for the assembly of thin filament lattice during development. The specific aims are: (1) to characterize nebulin isoforms in a wide range of skeletal muscles of fetal, neonatal and adult rabbits and rats. Nebulin expression will be compared and contrasted with the transition of other myofilament proteins such as titin, myosin, C-protein, actin, tropomyosin, troponin, alpha-actin, Cap Z and tropomodulin; (2) to identify and prepare isoform-specific and pan anti-nebulin antibodies. Epitope profiles of monoclonal anti-nebulin will be characterized for each isoform. Anti-peptide antibodies will be produced to common and variable regions of nebulin sequences; (3) to correlate the size of nebulin isoforms and the length of actin filaments in selected fetal, neonatal and adult skeletal muscles of rabbit and rats. Scanning transmission electron microscopy and mass spectroscopy will be used to determine the size and contour length of nebulin isoforms. Immunolocalization of nebulin isoforms, actin, tropomyosin, tropomodulin and cap Z will be used to determine the cellular distribution and length and span of thin filaments; and (4) to characterize the molecular basis of nebulin isoform diversity. Developmentally regulated expression of nebulin exons will be determined by polymerase chain reaction and nucleotide sequencing. The proposed research would define the physiological function of nebulin and the developmental and physiological basis for its contribution to the structure, regulation, and assembly of thin filaments in skeletal muscles in healthy and diseased states.
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1 |
1998 — 2010 |
Wang, Kuan |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Giant Proteins, Slender Filaments and Elastic Cytoskeleton @ Arthritis, Musculoskeletal, Skin Dis
Titin and nebulin are unprecedented giant proteins discovered and named in this laboratory. Our recent work has focused on the roles of titin and nebulin in the structure, regulation, mechanics and assembly of sarcomeres in striated muscles. We have employed a multidisciplinary approach that draws on methodologies and concepts of biochemistry, biophysics, cell and molecular biology, and physiology. (A) Titin: A primary theme of our research is the understanding of the structural and physiological roles of titin, a family of giant structural proteins that constitute an elastic matrix in the striated muscle sarcomeres and some nonmuscle cells. Titin and the elastic matrix may play major physiological roles, including the genesis of long range elasticity, the maintenance of sarcomere stability, the assembly of nascent sarcomeres in developing muscle cells, and the assembly of myosin in the microfilaments in nonmuscle cells. The following questions of central importance are being addressed: 1. How do titin filaments respond to stretch and release? 2. What is the molecular basis of titin elasticity? 3. Do thin filaments modulate titin elasticity? 4. Does titin participate in the regulation of actin-myosin interactions? 5. How do the signaling capacity of titin and titin kinase unction in muscle? We are testing the hypothesis that reversible unfolding of ordered domains such as titin kinase and reversible unraveling of intrinsically disordered segments are two distinct and complementary mechanisms of titin elasticity and function. The elasticity of isolated full length titin and engineered fragments and analogous elastic molecules from skeletal and cardiac and flight muscles are being measured directly, as single molecules and as gel networks, with laser trap techniques, atomic force microscopy (AFM) and rheometric techniques. The conformation of titin domains and synthetic peptides are being studied by high resolution NMR and X-ray crystallography. Titin elasticity is being explained with theories of biopolymers combined with realistic and experimental structural features of titin domains. In the past two years, we have succeeded in revealing that the elastic PEVK segment of titin is modular in construction, display repeating proline II helix /coil conformational motifs and most importantly, interact with thin filaments in a calcium/S100 sensitive manner. Our observations established the conformational basis of titin PEVK elasticity and identified PEVK as a site of interfilament interaction and that S100 or calcium sensor proteins regulate interaction and titin elasticity. Moreover, we discovered that the titin PEVK segment is a giant force sensing molecule of the SH3 signaling pathways. High resolution structures of a homopolymer of a PEVK module in solution and in gel phase has been determined by NMR techniques in collaboration with Prof. Wittebort at University of Louisville. The extensive differential splicing of titin PEVK exons in various human heart muscles was also elucidated. The PEVK domain is an intrinsically disordered protein with short-range structure. AFM measurements on PEVK suggested that its elasticity was driven in part by salt bridges. NMR data demonstrated the existence of specific salt bridges. Using the NIH Biowulf cluster, we applied molecular modeling techniques to understand dynamic interactions at the atomic level. TheMD and SMD simulations have shown further that the manifold of ionic interactions constantly evolves throughout the stretching. These new insights into the behavior of salt bridges in titin PEVK may provide insights in designing elastic polyampholytes as biomaterials for nanotechnology and tissue engineering. These simulations also help us to understand how ligand binding can be regulated by force and only bind when the protein is stretched to unmask SH3 binding sites. We are also designing a new type of instrrument to measure ligand binding and protein-protein interactions as proteins are being stretched. This new instrument will allow us to investigate directly how signaling protein interactions respond to mechanical stress. (B) Nebulin: Nebulin is the second largest protein ( 0.7 MDa) in the skeletal muscle sarcomere. It acts as a ruler for thin filament length and may act as a calcium-linked regulatory protein that is distinct from the classic troponin/tropomyosin system. In muscle development, nebulin is thought to expedite the maturation process of the thin filament assembly. Mutation in the nebulin gene leads to the disease nemalin myopathy, which causes muscle weakness and early death. The following questions are being addressed: 1. How does nebulin regulate the length of thin filaments? 2. How does nebulin regulate actin-myosin interaction? 3. What role does the nanomechanics of nebulin play in muscle function The length, mass and sequence of nebulin size isoforms have been compared with thin filament lengths of a wide range of skeletal muscles in developing and adult muscles. Together with the effect of over expression of nebulin domains on myofibrillogenesis in cultured skeletal muscle cells, we concluded that both the number of nebulin repeats and the specific N-terminal and C-terminal termination sequences are necessary to give rise to uniform thin filament length distribution within each sarcomere. In the past two years, we have successfully purified native full length nebulin from skeletal muscles and tested its effect on the length distribution of polymerizing or performed actin filaments. The measurement of actin filment length unfortunately was complicated by the gelling of actin filaments by the multivalent actin binding sites on each nebulin molecule. To alleviate this roblem, we are designing a new nanotechnology-based platform to measure the effect of nebulin on actin filament length distribution on a nanopatterned substrate that allows parallel actin filaments to elongate in the same direction and polarity without gelation. The kinetics of nebulin on ATPase activities is being studied by stop flow methods to define plausible molecular mechanisms of action. The influence of nebulin and calcium/calmodulin on the sliding velocity of actin over myosin is being investigated to reveal the effect of nebulin on mechanical coupling of myosin motors. We are searching for other potential calcium mediators by affinity chromatography and blotting. We are particularly interested in the possible involvement of S100 proteins as well as the kinase/phosphotase that phosphorylate nebulin as physiological effectors of actin/myosin interaction. We measured the nanomechncial properties of full-length native nebulin with the AFM. These required the implementation of the use of high affinity sitespecific antibodies pairwise to stretch nebulin between the nebulin;the use nof protein resistant self-assembled monolayers to prevent non-specific tip-surface interaction, a new method of sorting out single vs. multiple molecular stretching and the adaption of a powerful empirical mode decomposition (HHT) method to identify force-generating events. The most unique and unexpected finding is that nebulin must be stretched long enough to wrap around and match the peroriodicity of F-actin. We believe that these studies of full length nebulin and nebulin fragments will lead to a deeper understanding of how nebulin interacts with actin, myosin and other cellular components in the multiple roles it plays in muscle function. These results and the technology platform can also be applied to other members of the nebulin family, such as NRAP and cortactin which plays a role in muscle assemblyin muscle assemblyin muscle assembly, cell motility and hin muscle assembly, cell motility and has been implicated in muscle diseases and metastasis of cancer.
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0.916 |
2007 — 2010 |
Wang, Kuan |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Mechanical Biology and Muscle Development @ Arthritis, Musculoskeletal, Skin Dis
Our long term goal is to understand how contractile machinery of muscle cells assembles during development, how it dissembles during remodeling of muscle tissues, how external force and extracellular matrices affect gene expression and muscle structure and function, how the force is transmitted via the Z bands of the sarcomere, and how muscles malfunction in nemaline myopathy and other muscle diseases. The cytoplasm of striated muscle cells contains, besides actin and myosin filaments, contains at least two interconnected lattices. An intermediate filament lattice envelops and links all sarcomeres to the membrane skeleton, mitochondria, nuclei, and sarcoplasmic reticulum. Inside the sarcomere, a cytoskeletal matrix consisted of a set of elastic titin filaments and a set of inextensible nebulin filaments provides structural continuity. Both lattices generate restoring force. Active force and elastic force are transmitted through specialized anchor structures of the sarcomere. 1: Structure and function of the Z bands: One important stress-bearing structure is the Z line, a dense and narrow structure that anchors and organizes four major filaments: actin, titin, nebulin and desmin filaments. The Z lines play important roles in the structural organization of sarcomere, the transmission of mechanical forces as well as the stress signaling pathways. We are addressing the following questions: 1. what are the roles of titin, nebulin (skeletal muscles), nebulette (a nebulin-like protein in the heart) in the assembly and integrity of the Z line in vertebrate muscle? 2. What are the composition and structure of the unusually broad Z line of sonic muscle of Midshipman fish? 3. What are the roles of protein kinases, nonmuscle myosin and other signaling proteins in the function of the Z lines? 4. What is its relationship to the anomalous nemaline rod Z bodies found in aging heart muscle, in diseased skeletal muscle known as nemaline myopathy? (2) The roles of Xin family of proteins: In our search for binding partners of nebulin by yeast two-hybrid, we discovered that nebulin-SH3 binds to a large proline-rich region of human and mouse Xin protein, an adherens junction protein required for cardiac morphogenesis and looping (Wang, et al, 1999, Sinn, et al, 2002). Further characterization of this binding partner indicated that it was a heretofore unknown member of the Xin family that consists of two genes: aXin (first described by Jim Lin at Iowa) and bXin. Both aXin and bXin proteins have similar domain organization with an N-terminal repeat domain and a proline-rich domain. We are investigating the function of bXin in human and mouse. The human bXin (Chr. 2, 371 kb, 15 kb of 15 exons) and mouse bXin (Chr. 2, 330 kb, 12 kb of 13 exons) are expressed as complex, alternatively-spliced transcripts (7 for human bXin;5 for mouse bXin). One major product of the bXin gene is the bXin protein consisting of 3000 residues, with 94% being encoded by one long exon. Other alternatively-spliced variants exclude this exon and incorporate a LIM domain. Exon 7 and 13 of human bXin gene and exon 5 and 11 of mouse bXin gene are muscle-specific. The expression of bXin proteins is high in skeletal muscle and heart and low in lung and testis. bXin protein expression increases after differentiation in C2C12 myoblasts and after birth in mouse skeletal and heart muscle. In cultured mouse embryonic cardiomyocytes, bXin is localized at the stress fibers connecting myofibrils to focal adhesion and non-striated region along myofibrils. bXin appears to be important in the myosin isoform switching from nonmuscle myosin to sarcomeric myosin during myofibrillogenesis. Loss of bXin transcript and protein, by siRNA knock down technique, resulted in aberrant formation of myofibrils and caused the formation of extensive membrane protrusions. (3): Muscle assembly and disassemmbly in muscle cells subjected to external force: Since muscle cells both generate force internally and respond to force externally, we are investigating the cellular responses of muscle cells mechanical stress and how they alter their gene expression and programs of assembly and disassembly. The distribution of titin, nebulin, nebulette, Xin proteins and nonmuscle myosin IIA and IIB and into the myofibrils and the Z lines are being studied with siRNA knock down techniques and fluorescence localization techniques in live cells in culture. The muscle cells are subjected to cyclic strains on elastic substrate of defined stiffness. (4): The role of nebulin in nemaline myopathy animal model: We are collaborating with Dr. Ju Chen at UCSD to characterize the muscle on the further characterization of striated muscles in the nebulin knock out mice that they generated. We are determining determined the thin filament length distribution in skeletal muscles by electron and immunoelectron microscopy to assess the distribution of actin/tropomyosin/troponin, capping proteins such as tropomodulin, myopallidin and CapZ along the thin filaments as well as the glycogen granule proteins. We also plan to study the calcium activation of contraction of these muscles subjected to cyclic mechanical activity in vivo and as isolated muscle fibers to evaluate their extent of mechanical damage and their force pCa and force ATPase curves. Additionally, the rate of formation of nemaline rods will be monitored to assess the effect of external force on the nemaline rod formation. We aim to understand the molecular basis of the formation of nemaline rods and whether the formation of inclusion bodies in muscle diseases such as nemaline myopathy is a coping mechanism to deal with weakened sarcomeres or force transmission.
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0.916 |
2007 — 2018 |
Wang, Kuan [⬀] |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Molecular and Cellular Mechanisms of Experience-Dependent Cortical Processing @ National Institute of Mental Health
Our lab produced Activity-Regulated Cytoskeleton-Associated (ARC) knockout mice to be used in a collaborative project studying how the loss of the ARC protein may alter the effects of sensory experience or deprivation in the visual cortex (V1), McCurry et al (Nature Neuroscience 2010). The data indicate that the loss of ARC protein leads to an absence of ocular dominance plasticity in response to brief and extended monocular deprivation as well as open-eye potentiation, suggesting that ARC is necessary for deprived-eye depression that normally takes place after monocular deprivation, as seen in the normal mice. The observed deficits occurred in the absence of major changes in visual response properties, since ARC -/- mice exhibited normal visual acuity and retinotopic organization. ARC may play a role in the refinement of response properties such as the reduction or removal of weaker inputs and the potentiation of stronger inputs giving rise to sharpened receptive field properties. This study discovered that when the visual cortex is missing the ARC gene, sensory experience or deprivation cannot alter visual cortical functions. Our lab continues to investigate the mechanisms by which experience-induced molecular changes impact on cortical processing of information, with a particular focus on prefrontal cortical circuits. Normal executive function in goal-directed behavior depends on the prefrontal cortex, and functional brain imaging studies have revealed altered prefrontal activity in response to cognitive challenges in schizophrenia patients. However, the mechanisms by which specific genetic risk factors and behavioral experiences may influence the functional cellular architecture and the developmental trajectory of prefrontal cortical circuits remain largely unknown. We have developed techniques to identify specific neurons with experience-dependent gene expression changes in prefrontal circuits, and combined those techniques with electrophysiological and calcium imaging methods to determine the functional contributions of those neurons to prefrontal circuit outputs. In addition, we have developed chronicle in vivo imaging techniques to track experience-dependent molecular changes in live animals while the animals learn new tasks, which may help to elucidate the processes by which neuronal ensembles in the prefrontal cortes adapt to different environments and situations. Our group continues to investigate the coupling mechanisms between neuronal activity and plasticity-related gene expression in cortical circuits, using both molecular genetic and optical imaging tools. Particularly, we are examining whether the induction of activity-dependent gene expression is modified under the direct influence of specific neuromodulators that are associated with the motivational or emotional relevance of a given behavioral experience. Finally, we continue to apply in vivo imaging and optogenetic techniques to study the functional plasticity of prefrontal circuits in normal adolescent and adult animals, and prefrontal dysfunctions in the mouse models of psychiatric disorders as developed by the other research groups in the Genes, Cognition and Psychosis Program. Those studies may help to monitor the development of abnormal cortical circuits in real time, and elucidate the interactions of genetic risk factors with environmental and social stressors.
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0.975 |
2020 — 2021 |
Wang, Kuan Hong [⬀] |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Bridging the Translational Divide From Cells to Patients: Toward Reliable Neuromarkers of Batten Disease @ University of Rochester
PROJECT SUMMARY Neuronal Ceroid Lipofuscinoses (NCLs) are a group of neurodevelopmental diseases categorized as autosomal recessive lysosomal storage disorders. Clinical features include retinopathy, intracellular accumulation of lysosomal ceroid and lipofuscin, seizures, motor decline and dementia. CLN3 disease (Juvenile Neuronal Ceroid Lipofuscinosis, JNCL) is one of the most common types of NCL, and results from mutations in the CLN3 gene on chromosome 16. Individuals with the CLN3 mutation show a consistent decline in cognitive functioning and verbal intellectual abilities over the course of later childhood and early adolescence. The precise neuropathological bases of this decline are not yet well understood and objective neurologic biomarkers (neuromarkers) of disease progression are not currently available. Yet, our preliminary data indicate that a good candidate for a biomarker of CLN3 disease that tracks with disease severity can be identified using high-density electroencephalography (EEG) in the context of auditory mismatch negativity experiments. Using these methods we have been able to show consistent declines in the P1 component of the auditory evoked response as CLN3 disease severity increases. Here we propose to further develop our understanding of auditory processing in children and young adults with CLN3 disease with a focus on validating a biomarker of the disease. Simultaneously we propose to use a mouse model of CLN3 disease with the same genetic mutation to identify an endophenotype that is shared in both patients and mice. We will also test whether the accumulation of autofluorescent lipopigments preferentially occur in specific subsets of interneurons and predict their subsequent loss as well as the hypertrophy of remaining interneurons. This three-pronged, convergence of techniques and approaches in both species has the potential to yield unique opportunities to understand the underlying neurophysiology of CLN3 mutations and their impact on auditory processing, as well as encourage testing of future treatments for CLN3 disease first in mice and secondarily in patients. This innovative approach, tying the patient neurophysiological markers to identical measures in the murine model of the disease will permit improved and more efficient pre-clinical development of novel therapeutics, improved measurement of disease progression in clinical trials, and with these advances, lead to improved outcomes for patients with CLN3 mutations.
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0.916 |
2021 |
Conwell, Yeates (co-PI) [⬀] Lin, Feng Vankee Wang, Kuan Hong (co-PI) [⬀] |
U24Activity Code Description: To support research projects contributing to improvement of the capability of resources to serve biomedical research. |
Network For Emotional Wellbeing and Brain Aging (New Brain Aging) @ University of Rochester
Project summary: Early evidence indicates an association between emotional (eudaimonic and hedonic) well- being (EWB) and underlying brain processes, and that those processes change with both normal and pathological brain aging. However, the nature of these associations, the mechanisms by which EWB and its component domains change with brain aging, and how those changes may be associated with common neuropathologies like Alzheimer?s disease and related dementias (ADRD), are largely unexplored. The objective of the Network for Emotional Well-being and Brain Aging (NEW Brain Aging) is to address priority area #2 ? mechanistic research on the role of emotional well-being in health ? of RFA-AT-20-003, identifying and testing mechanisms by which brain aging influences EWB and how EWB may impact risk for and progression of ADRD. Synthesizing human and animal literature, our premise is that relationships between EWB and ADRD are bidirectional ? normal and pathological changes in aging brain influence EWB and EWB contributes to brain health and illness, such as ADRD. Further, we hypothesize that the relationships between EWB and the brain in older adults reflect aging-associated heterogeneity of brain (i.e., resilience, typical aging, or neuropathological changes), and that they are mediated by processes of appraisal and adaptation. To do so we will form a national, transdisciplinary collaborative that includes investigators representing research expertise in human and animal neuroimaging, stress regulation, ADRD research, EWB, and computational/quantitative methods. With direction of an Executive Committee and guided by an External Advisory Committee, NEW Brain Aging will undertake these specific aims: (1) To build an inter-university and transdisciplinary collaborative of senior and junior investigators interested in brain, aging, and EWB research from both human and animal fields; (2) To form a series of time-limited workgroups with relevant experts in the field, each working over 1-2 years, to establish priorities for NEW Brain Aging activities in each of three general areas: (a) brain mechanism research of EWB; (b) research of EWB?s impact on ADRD; and (c) translational animal and cross-species brain research methods; (3) To coalesce and coordinate resources to ensure the rigor and reproducibility of the brain-related EWB research; (4) To provide three types of pilot funding through rigorous peer review: (a) pilot research grants for junior faculty related to brain mechanism studies related to the relationship between EWB and dementia using human or animal models; (b) proof-of-concept grants for established investigators to test aspects related to EWB in their existing studies and cohorts of brain aging; (c) seed collaboration grants for cross-species brain research related to EWB; (5) To evaluate the network by tracking its activities, resource usage, and productivity, and conducting collaborative network density analysis throughout the 4-year grant; and (6) to ensure the wide dissemination of NEW Brain Aging products. By accomplishing these aims, we will strengthen a research network focusing on the brain mechanistic understanding of EWB and relationship between EWB and ADRD.
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