In JoVE (1)

Other Publications (20)

Articles by Scott Medler in JoVE

Other articles by Scott Medler on PubMed

Myofibrillar Protein Isoform Expression is Correlated with Synaptic Efficacy in Slow Fibres of the Claw and Leg Opener Muscles of Crayfish and Lobster

The Journal of Experimental Biology. Feb, 2002  |  Pubmed ID: 11893765

In the crayfish and lobster opener neuromuscular preparations of the walking legs and claws, there are regional differences in synaptic transmission even though the entire muscle is innervated by a single excitatory tonic motor neuron. The innervation of the proximal fibres produced larger excitatory postsynaptic potentials (EPSPs) than those of the central fibres. The amplitudes of the EPSPs in the distal fibres were intermediate between those of the proximal and central regions. These differences in EPSP amplitudes were correlated with differences in short-term facilitation between the three regions. When given a 10- or 20-pulse train of stimuli, the proximal fibres showed greater short-term facilitation initially, often followed by a maximization of short-term facilitation towards the end of a train. In contrast, the central fibres showed a linear increase in short-term facilitation throughout a stimulus train. The distal fibres showed intermediate short-term facilitation compared with the other two regions. Analysis of myofibrillar isoforms showed that levels of troponin-T(1) (TnT(1)), a 55 kDa isoform expressed in slow-tonic (S(2)) fibres, were correlated with synaptic properties. Proximal fibres had the highest levels of TnT(1), with lower levels in distal fibres; central fibres lacked TnT(1), which is characteristic of slow-twitch (S(1)) fibres. In addition, differences in troponin-I isoforms correlated with TnT(1) levels between the proximal, central and distal regions. The correlation between slow fibre phenotype and strength of innervation suggests a relationship between synaptic structure and expression of troponin isoforms.

Comparative Trends in Shortening Velocity and Force Production in Skeletal Muscles

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Aug, 2002  |  Pubmed ID: 12121850

Skeletal muscles are diverse in their properties, with specific contractile characteristics being matched to particular functions. In this study, published values of contractile properties for >130 diverse skeletal muscles were analyzed to detect common elements that account for variability in shortening velocity and force production. Body mass was found to be a significant predictor of shortening velocity in terrestrial and flying animals, with smaller animals possessing faster muscles. Although previous studies of terrestrial mammals revealed similar trends, the current study indicates that this pattern is more universal than previously appreciated. In contrast, shortening velocity in muscles used for swimming and nonlocomotory functions is not significantly affected by body size. Although force production is more uniform than shortening velocity, a significant correlation with shortening velocity was detected in muscles used for locomotion, with faster muscles tending to produce more force. Overall, the contractile properties of skeletal muscles are conserved among phylogenic groups, but have been significantly influenced by other factors such as body size and mode of locomotion.

Analysis of Myofibrillar Proteins and Transcripts in Adult Skeletal Muscles of the American Lobster Homarus Americanus: Variable Expression of Myosins, Actin and Troponins in Fast, Slow-twitch and Slow-tonic Fibres

The Journal of Experimental Biology. Oct, 2003  |  Pubmed ID: 12966047

Skeletal muscles are diverse in their contractile properties, with many of these differences being directly related to the assemblages of myofibrillar isoforms characteristic of different fibers. Crustacean muscles are similar to other muscles in this respect, although the majority of information about differences in muscle organization comes from vertebrate species. In the present study, we examined the correlation between myofibrillar protein isoforms and the patterns of myofibrillar gene expression in fast, slow-phasic (S(1)) and slow-tonic (S(2)) fibers of the American lobster Homarus americanus. SDS-PAGE and western blotting were used to identify isoform assemblages of myosin heavy chain (MHC), P75, troponin T (TnT) and troponin I (TnI). RT-PCR was used to monitor expression of fast and slow (S(1)) MHC, P75 and actin in different fiber types, and the MHC and actin levels were quantified by real-time PCR. Fast and slow fibers from the claw closers predominantly expressed fast and S(1) MHC, respectively, but also lower levels of the alternate MHC. By contrast, fast fibers from the deep abdominal muscle expressed fast MHC exclusively. In addition, slow muscles expressed significantly higher levels of actin than fast fibers. A distal bundle of fibers in the cutter claw closer muscle was found to be composed of a mixture of S(1) and S(2) fibers, many of which possessed a mixture of S(1) and S(2) MHC isoforms. This pattern supports the idea that S(1) and S(2) fibers represent extremes in a continuum of slow muscle phenotype. Overall, these patterns demonstrate that crustacean skeletal muscles cannot be strictly categorized into discrete fiber types, but a muscle's properties probably represent a point on a continuum of fiber types. This trend may result from differences in innervation pattern, as each muscle is controlled by a unique combination of phasic, tonic or both phasic and tonic motor nerves. In this respect, future studies examining how muscle phenotype correlates with innervation pattern may help account for variation in crustacean fiber types.

Two Fast-type Fibers in Claw Closer and Abdominal Deep Muscles of the Australian Freshwater Crustacean, Cherax Destructor, Differ in Ca2+ Sensitivity and Troponin-I Isoforms

Journal of Experimental Zoology. Part A, Comparative Experimental Biology. Jul, 2004  |  Pubmed ID: 15229869

One type of fast fiber and two types of slow (slow-twitch, S1 and slow-tonic, S2) fibers are found in decapod crustacean skeletal muscles that differ in contractile properties and myofibrillar protein isoform compositions. In this study the structural characteristics, protein isoform compositions, and Ca2+-activation properties of fast fibers in the claw closer (F1) and abdominal deep flexor (F2) muscles of Cherax destructor were analyzed. For comparison, myofibrillar protein isoform compositions of slow (long-sarcomere) fibers from claw and abdomen were also determined; our results indicate that the slow fibers in the claw closer were the slow-twitch (S1) type and those in the abdominal superficial flexor were primarily slow-tonic (S2) type. F1 fibers had shorter resting sarcomere lengths (2.93 microm in unstretched fibers and 3.06 microm in stretched fibers) and smaller fiber diameter (256 microm) than F2 fibers (sarcomere lengths 3.48 microm in unstretched and 3.46 microm in stretched; 747 microm diameter). Moreover, F1 fibers showed a narrower range in sarcomere lengths than F2 fibers (2.81 to 3.28 microm vs. 2.47 to 4.05 micro m in unstretched fibers). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the fast fibers from claw and abdomen differed in troponin-I composition; F1 fibers expressed two isoforms of troponin-I (TnI1 and TnI2) in approximately equal amounts, whereas F2 fibers expressed primarily TnI3 and lower levels of TnI1. F1 fibers were more sensitive to Ca2+, as shown by higher pCa values at threshold activation (pCa(10)=6.50+/-0.07) and at 50% maximum force (pCa(50)=6.43+/-0.07) than F2 fibers (pCa(10)=6.12+/-0.04 and pCa(50)=5.88+/-0.03, respectively). F1 fibers also had a greater degree of co-operativity in Ca2+ activation, as shown by a higher maximum slope of the force-pCa curve (n(Ca)=12.98+/-2.27 vs. 4.34+/-0.64). These data indicate that there is a greater fast fiber-type diversity in crustacean muscles than was previously supposed. Moreover, the differences in activation properties suggest that the TnI isoform composition influences the Ca2+ sensitivity of the contractile mechanism.

Fiber Polymorphism in Skeletal Muscles of the American Lobster, Homarus Americanus: Continuum Between Slow-twitch (S1) and Slow-tonic (S2) Fibers

The Journal of Experimental Biology. Jul, 2004  |  Pubmed ID: 15235004

In recent years, an increasing number of studies has reported the existence of single fibers expressing more than one myosin heavy chain (MHC) isoform at the level of fiber proteins and/or mRNA. These mixed phenotype fibers, often termed hybrid fibers, are currently being recognized as the predominant fiber type in many muscles, and the implications of these findings are currently a topic of great interest. In a recent study, we reported single fibers from the cutter claw closer muscle of lobsters that demonstrated a gradation between the slow-twitch (S1) and slow-tonic (S2) muscle phenotype. In the present study, we focused on S1 and S2 fibers from the superficial abdominal muscles of the lobster as a model to study the continuum among muscle fiber types. Complementary DNAs (cDNA) encoding an S2 isoform of myosin heavy chain (MHC) and an S2 isoform of tropomyosin (Tm) were isolated from the superficial abdominal flexor muscles of adult lobsters. These identified sequences were used to design PCR primers used in conjunction with RT-PCR and real-time PCR to measure expression levels of these genes in small muscle samples and single fibers. The relative expression of the corresponding S1 MHC and S1 Tm isoforms was measured in the same samples with PCR primers designed according to previously identified sequences. In addition, we measured the relative proportions of MHC, troponin (Tn) T and I protein isoforms present in the same samples to examine the correlation of these proteins with one another and with the MHC and Tm mRNAs. These analyses revealed significant correlations among the different myofibrillar proteins, with the S1 and S2 fibers being characterized by a whole assemblage of myofibrillar isoforms. However, they also showed that small muscle samples, and more importantly single fibers, existed as a continuum from one phenotype to another. Most fibers possessed mixtures of mRNA for MHC isoforms that were unexpected based on protein analysis. These findings illustrate that muscle fibers in general may possess a phenotype that is intermediate between the extremes of 'pure' fiber types, not only at the MHC level but also in terms of whole myofibrillar assemblages. This study supports and extends our recent observations of mixed phenotype fibers in lobster claw and leg muscles. The existence of single fiber polymorphism in an invertebrate species underscores the generality of the phenomenon in skeletal muscles and emphasizes the need for an understanding of the proximal causes and physiological consequences of these intermediate fiber types.

Eyestalk Ablation Has Little Effect on Actin and Myosin Heavy Chain Gene Expression in Adult Lobster Skeletal Muscles

The Biological Bulletin. Apr, 2005  |  Pubmed ID: 15837962

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.

Myofibrillar Gene Expression in Differentiating Lobster Claw Muscles

Journal of Experimental Zoology. Part A, Ecological Genetics and Physiology. May, 2007  |  Pubmed ID: 17377955

Lobster claw muscles undergo a process of fiber switching during development, where isomorphic muscles containing a mixture of both fast and slow fibers, become specialized into predominantly fast, or exclusively slow, muscles. Although this process has been described using histochemical methods, we lack an understanding of the shifts in gene expression that take place. In this study, we used several complementary techniques to follow changes in the expression of a number of myofibrillar genes in differentiating juvenile lobster claw muscles. RNA probes complementary to fast and slow myosin heavy chain (MHC) mRNA were used to label sections of 7th stage (approximately 3 months old) juvenile claw muscles from different stages of the molt cycle. Recently molted animals (1-5 days postmolt) had muscles with distinct regions of fast and slow gene expression, whereas muscles from later in the molt cycle (7-37 days postmolt) had regions of fast and slow MHC expression that were co-mingled and indistinct. Real-time PCR was used to quantify several myofibrillar genes in 9th and 10th stages (approximately 6 months old) juvenile claws and showed that these genes were expressed at significantly higher levels in the postmolt claws, as compared with the intermolt and premolt claws. Finally, Western blot analyses of muscle fibers from juvenile lobsters approximately 3 to 30 months in age showed a shift in troponin-I (TnI) isoform expression as the fibers differentiated into the adult phenotypes, with expression of the adult fast fiber TnI pattern lagging behind the adult slow fiber TnI pattern. Collectively, these data show that juvenile and adult fibers differ both qualitatively and quantitative in the expression of myofibrillar proteins and it may take as much as 2 years for juvenile fibers to achieve the adult phenotype.

Muscle-specific Calpain is Localized in Regions Near Motor Endplates in Differentiating Lobster Claw Muscles

Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology. Nov, 2007  |  Pubmed ID: 17827046

Calpains are Ca2+-dependent proteinases that mediate protein turnover in crustacean skeletal muscles. We used an antibody directed against lobster muscle-specific calpain (Ha-CalpM) to examine its distribution in differentiating juvenile lobster claw muscles. These muscles are comprised of both fast and slow fibers early in development, but become specialized into predominantly fast or exclusively slow muscles in adults. The transition into adult muscle types requires that myofibrillar proteins specific for fast or slow muscles to be selectively removed and replaced by the appropriate proteins. Using immunohistochemistry, we observed a distinct staining pattern where staining was preferentially localized in the fiber periphery along one side of the fiber. Immunolabeling with an antibody directed against synaptotagmin revealed that the calpain staining was greatest in the cytoplasm adjacent to synaptic terminals. In complementary analyses, we used sequence-specific primers with real-time PCR to quantify the levels of Ha-CalpM in whole juvenile claw muscles. These expression levels were not significantly different between cutter and crusher claws, but were positively correlated with the expression of fast myosin heavy chain. The anatomical localization of Ha-CalpM near motor endplates, coupled with the correlation with fast myofibrillar gene expression, suggests a role for this intracellular proteinase in fiber type switching.

Frequency-dependent Power Output and Skeletal Muscle Design

Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology. Mar, 2009  |  Pubmed ID: 19101645

Cyclically contracting muscles provide power for a variety of processes including locomotion, pumping blood, respiration, and sound production. In the current study, we apply a computational model derived from force-velocity relationships to explore how sustained power output is systematically affected by shortening velocity, operational frequency, and strain amplitude. Our results demonstrate that patterns of frequency dependent power output are based on a precise balance between a muscle's intrinsic shortening velocity and strain amplitude. We discuss the implications of this constraint for skeletal muscle design, and then explore implications for physiological processes based on cyclical muscle contraction. One such process is animal locomotion, where musculoskeletal systems make use of resonant properties to reduce the amount of metabolic energy used for running, swimming, or flying. We propose that skeletal muscle phenotype is tuned to this operational frequency, since each muscle has a limited range of frequencies at which power can be produced efficiently. This principle also has important implications for our understanding muscle plasticity, because skeletal muscles are capable of altering their active contractile properties in response to a number of different stimuli. We discuss the possibility that muscles are dynamically tuned to match the resonant properties of the entire musculoskeletal system.

Skeletal Muscle Fiber Types in the Ghost Crab, Ocypode Quadrata: Implications for Running Performance

The Journal of Experimental Biology. Mar, 2009  |  Pubmed ID: 19218519

Ghost crabs possess rapid running capabilities, which make them good candidates for comparing invertebrate exercise physiology with that of more extensively studied vertebrates. While a number of studies have examined various aspects of running physiology and biomechanics in terrestrial crabs, none to date have defined the basic skeletal muscle fiber types that power locomotion. In the current study, we investigated skeletal muscle fiber types comprising the extensor and flexor carpopodite muscles in relation to running performance in the ghost crab. We used kinematic analyses to determine stride frequency and muscle shortening velocity and found that both parameters are similar to those of comparably sized mammals but slower than those observed in running lizards. Using several complementary methods, we found that the muscles are divided into two primary fiber types: those of the proximal and distal regions possess long sarcomeres (6.2+/-2.3 microm) observed in crustacean slow fibers and have characteristics of aerobic fibers whereas those of the muscle mid-region have short sarcomeres (3.5+/-0.4 microm) characteristic of fast fibers and appear to be glycolytic. Each fiber type is characterized by several different myofibrillar protein isoforms including multiple isoforms of myosin heavy chain (MHC), troponin I (TnI), troponin T (TnT) and a crustacean fast muscle protein, P75. Three different isoforms of MHC are differentially expressed in the muscles, with fibers of the mid-region always co-expressing two isoforms at a 1:1 ratio within single fibers. Based on our analyses, we propose that these muscles are functionally divided into a two-geared system, with the aerobic fibers used for slow sustained activities and the glycolytic mid-region fibers being reserved for explosive sprints. Finally, we identified subtle differences in myofibrillar isoform expression correlated with crab body size, which changes by several orders of magnitude during an animal's lifetime.

Gene Looping is Conferred by Activator-dependent Interaction of Transcription Initiation and Termination Machineries

The Journal of Biological Chemistry. Sep, 2009  |  Pubmed ID: 19602510

Gene looping juxtaposes the promoter and terminator regions of RNA polymerase II-transcribed genes in yeast and mammalian cells. Here we report an activator-dependent interaction of transcription initiation and termination factors during gene looping in budding yeast. Chromatin analysis revealed that MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during activated transcription. Looping was nearly abolished in the absence of transcription activators Met28, Ino2, and Gal4 of MET16, INO1, and GAL1p-BUD3 genes, respectively. The activator-independent increase in transcription was not accompanied by loop formation, thereby suggesting an essential role for activators in gene looping. The activators did not facilitate loop formation directly because they did not exhibit an interaction with the 3' end of the genes. Instead, activators physically interacted with the general transcription factor TFIIB when the genes were activated and in a looped configuration. TFIIB cross-linked to both the promoter and the terminator regions during the transcriptionally activated state of a gene. The presence of TFIIB on the terminator was dependent on the Rna15 component of CF1 3' end processing complex. Coimmunoprecipitation revealed a physical interaction of Rna15 with TFIIB. We propose that the activators facilitate gene looping through their interaction with TFIIB during transcriptional activation of genes.

Relative Proportions of Hybrid Fibres Are Unaffected by 6 Weeks of Running Exercise in Mouse Skeletal Muscles

Experimental Physiology. Jan, 2010  |  Pubmed ID: 19748966

Hybrid muscle fibres co-expressing two or more myosin heavy chain (MHC) isoforms represent a significant proportion of fibres in many muscles, but the prevalence and precise composition of these fibres varies significantly among muscles and animal species. In the present study, we used a forced running protocol for 6 weeks to determine the effects of running exercise on the relative proportion of hybrid muscle fibre types in mouse muscles. In the course of this experiment, we also determined the relative proportions of these fibres in several different skeletal muscles, since data about hybrid fibres in the mouse are sparse. We found that the proportions of hybrid fibres in mouse muscles varied significantly among specific muscles (2-25%), but these proportions were unaffected by 6 weeks of forced running exercise. In contrast, weight-bearing muscles significantly increased in mass in response to running. These data suggest that hybrid muscle fibres are relatively refractory to the effects of moderate exercise and represent a stable phenotype in normal mouse muscles. The precise nature and physiological function of these fibres remain incompletely understood, but it is clear that hybrid fibres represent a common phenotype in many muscles.

Myostatin from the American Lobster, Homarus Americanus: Cloning and Effects of Molting on Expression in Skeletal Muscles

Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology. Dec, 2010  |  Pubmed ID: 20696264

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S₁) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

The Continuum of Hybrid IIX/IIB Fibers in Normal Mouse Muscles: MHC Isoform Proportions and Spatial Distribution Within Single Fibers

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Dec, 2010  |  Pubmed ID: 20861278

Although skeletal muscle fiber types are often defined as belonging to discrete categories, many muscles possess fibers with intermediate phenotypes. These hybrid fiber types can be identified by their expression of two or more myosin heavy chain (MHC) isoforms within the same single fiber. In mouse muscles, the most common hybrid fibers are those coexpressing the IIX and IIB MHC isoforms. In the present study, we focused on these IIX/IIB fibers from normal mouse muscles to determine the relative proportions of MHC isoforms at both the protein and mRNA levels and to examine the longitudinal distribution of isoforms within single fibers. We found that IIX/IIB hybrids represent ∼25 and 50% of the fibers in the mouse tibialis anterior and brachioradialis, respectively. The relative proportion of the IIX and IIB isoforms in these fibers spans a continuum, from predominantly IIB-like hybrids to IIX-like hybrids. Quantitative assessment of mRNA levels using real-time PCR from single fibers indicated that IIB expression dominated over IIX expression in most fibers and that a general correlation existed between mRNA isoform levels and MHC protein content. However, the match between mRNA levels and protein content was not precise. Finally, we measured MHC isoform proportions in adjacent fiber segments and discovered that ∼30% of hybrids possessed significant differences in isoform content along their length. In some instances, the muscle fiber type as defined by MHC content changed completely along the length of a fiber. This pattern of asymmetrical MHC isoform content along the length of single fibers suggests that the multiple myonuclei of a muscle fiber may express distinct myofibrillar isoforms in an uncoordinated fashion.

Evidence for a Complex of Transcription Factor IIB with Poly(A) Polymerase and Cleavage Factor 1 Subunits Required for Gene Looping

The Journal of Biological Chemistry. Sep, 2011  |  Pubmed ID: 21835917

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

Analysis of Interactions Between Genomic Loci Through Chromosome Conformation Capture (3C)

Current Protocols in Cell Biology. Sep, 2012  |  Pubmed ID: 22968842

Genome architecture plays a significant role in the regulation of DNA-based cellular processes such as transcription and recombination. The successful accomplishment of these processes involves coordinated interaction of DNA elements located at a distance from each other. The 'Chromosome Conformation Capture' (3C) assay is a convenient tool for identification of physical association between spatially separated DNA elements in a cell under physiological conditions. The principle of 3C is to convert physical chromosomal interactions into specific DNA ligation products, which are then detected by PCR. The 3C protocol was originally used to identify long-range, stable chromosomal interactions in yeast. Here we describe a modified 3C procedure that can detect transient, short-range interactions of DNA elements separated by a distance of less than 700 bp. This method has been successfully used to detect dynamic interaction of transcription regulatory elements in yeast and can be used for detecting similar interactions of other genomic regions.

Hybrid Fibers Transform into Distinct Fiber Types in Maturing Mouse Muscles

Cells, Tissues, Organs. 2013  |  Pubmed ID: 24192720

The role of hybrid fibers as intermediates in fiber type transformations is not completely understood. In some cases hybrids are clearly transitional fibers changing from one type to another, but in others they represent phenotypically stable fibers in normal muscles. In the current study, our goal was to understand the fate of hybrid fibers in fiber type transitions that take place during muscle maturation. Previous studies have reported high proportions of hybrid fibers during postnatal development, but few have followed the fate of these fibers past the time of weaning. We quantified proportions of hybrid fibers in three different mouse skeletal muscles from newly weaned to 6-month-old mice. Hybrid fibers were very prevalent in the brachioradialis (BR) and tibialis anterior (TA) muscles from newly weaned mice, where they constituted 50 and 40% of the fibers, respectively. These hybrids declined steadily to about 15-30% over the next several months. In the soleus muscle the proportion of hybrids did not change, but constituted approximately 20% of fibers. The reduction in IIX/IIB hybrids resulted from different processes in the BR and the TA. In the BR, the reduction was coincident with an increase in type IIX fibers. In the TA, the number of IIX/IIB hybrids was inversely correlated with the proportion of IIB fibers. These patterns reveal that the role of hybrid fibers as intermediates in muscle development is complex. Some hybrid fibers in maturing muscles represent transitional fiber types, while others are phenotypically stable. Moreover, the fate of transitional fibers may be distinct among similar fiber types within different muscles.

Measuring Dynamic Kidney Function in an Undergraduate Physiology Laboratory

Advances in Physiology Education. Dec, 2013  |  Pubmed ID: 24292917

Most undergraduate physiology laboratories are very limited in how they treat renal physiology. It is common to find teaching laboratories equipped with the capability for high-resolution digital recordings of physiological functions (muscle twitches, ECG, action potentials, respiratory responses, etc.), but most urinary laboratories still rely on a "dipstick" approach of urinalysis. Although this technique can provide some basic insights into the functioning of the kidneys, it overlooks the dynamic processes of filtration, reabsorption, and secretion. In the present article, we provide a straightforward approach of using renal clearance measurements to estimate glomerular filtration rate, fractional water reabsorption, glucose clearance, and other physiologically relevant parameters. The estimated values from our measurements in laboratory are in close agreement with those anticipated based on textbook parameters. For example, we found glomerular filtration rate to average 124 ± 45 ml/min, serum creatinine to be 1.23 ± 0.4 mg/dl, and fractional water reabsorption to be ∼96.8%. Furthermore, analyses for the class data revealed significant correlations between parameters like fractional water reabsorption and urine concentration, providing opportunities to discuss urine concentrating mechanisms and other physiological processes. The procedures outlined here are general enough that most undergraduate physiology laboratory courses should be able to implement them without difficulty.

Diet-induced Obesity Alters Skeletal Muscle Fiber Types of Male but Not Female Mice

Physiological Reports. Jan, 2014  |  Pubmed ID: 24744883

Skeletal muscles are highly plastic tissues capable dramatic remodeling in response to use, disuse, disease, and other factors. Growing evidence suggests that adipose tissues exert significant effects on the basic fiber-type composition of skeletal muscles. In the current study, we investigated the long-term effects of a high-fat diet and subsequent obesity on the muscle fiber types in C57 BLK/6J mice. Litters of mice were randomly assigned to either a high-fat diet or a control group at the time of weaning, and were maintained on this diet for approximately 1 year. Single fibers were harvested from the soleus and plantaris muscles, and fiber types were determined using SDS-PAGE. The high-fat diet mice were significantly heavier than the control mice (39.17 ± 2.7 g vs. 56.87 ± 3.4 g; P < 0.0003), but muscle masses were not different. In male mice, the high-fat diet was associated with a significantly lower proportion of slow, type I fibers in the soleus muscle (40.4 ± 3.5% vs. 29.33 ± 2.6%; P < 0.0165). Moreover, the proportion of type I fibers in the soleus of male mice was inversely proportional to the relative fatness of the male mice (P < 0.003; r (2) = 0.65), but no association was observed in female mice. In male mice, the decline in type I fibers was correlated with an increase in type I/IIA hybrid fibers, suggesting that the type I fibers were transformed primarily into these hybrids. The reported trends indicate that type I fibers are most susceptible to the effects of obesity, and that these fiber-type changes can be sex specific.

Gene Looping Facilitates TFIIH Kinase-mediated Termination of Transcription

Scientific Reports. Aug, 2015  |  Pubmed ID: 26286112

TFIIH is a general transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of transcription is well established. Here we report a novel role of Kin28 in the termination of transcription. We show that RNAPII reads through a termination signal upon kinase inhibition. Furthermore, the recruitment of termination factors towards the 3' end of a gene was compromised in the kinase mutant, thus confirming the termination defect. A concomitant decrease in crosslinking of termination factors near the 5' end of genes was also observed in the kinase-defective mutant. Simultaneous presence of termination factors towards both the ends of a gene is indicative of gene looping; while the loss of termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the termination of transcription in budding yeast.

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