Molecular, Genetics, and Biochemical Studies on Troponin I and Tropomyosin in the Regulation of Muscle Contraction Open Access

Barnes, Dawn Elizabeth (2016)

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The nematode Caenorhabditis elegans uses the striated muscles of its body wall for locomotion. The movement is achieved through the collective shortening of sarcomeres on one side of the body (contraction) and simultaneous lengthening of sarcomeres on the opposite side of the body (relaxation). Muscle contraction results from shortening of sarcomeres achieved by interactions between thin filament containing F-actin and thick filament with bipolar arrangement of myosin motors. The myosin motors are constitutively-active and thus the control between actomyosin interactions is through regulatory component of the thin filaments. Mutations of sarcomere proteins often cause uncoordinated ("unc") phenotypes. However, there was no techniques available to analyze kinetics of worm muscle contraction and relaxation in a quantitative manner. We have utilized optogenetically-induced activation of muscle contraction as a novel technique to study the roles of sarcomere proteins in locomotion and we found functional relationships between proteins within the sarcomere. Among these proteins, tropomyosin is important in regulating actomyosin interactions in cooperation with the troponin complex. We identified a novel high molecular weight tropomyosin isoform (LEV-11O) that is the first LEV-11 protein including alternative Exon 7 (7a). We found that LEV-11O has similar biochemical properties to other tropomyosin isoforms and is the major isoform in the body wall muscles of the head-region. The position of tropomyosin in the thin filament is regulated by the troponin complex. Within the troponin complex, troponin I is sufficient to stabilize tropomyosin in a position to inhibit myosin binding. Through sequence analysis we conclude that the core domains of troponin I are conserved across the bilaterian phyla we examined, however, the N- and C-terminal extensions are variable. The N-terminal extension is present in all protostome troponin I and chordate cardiac troponin I, however this region is absent from chordate skeletal muscle isoforms. Through in vivo studies, we observed that the N-terminal extension is important in the coordinated muscle contractility important for the sinusoidal locomotion of C. elegans. Our work has contributed to the understanding of how different isoforms of thin filament proteins can be important in regulating the activities of different types of muscle.

Table of Contents

1 Introduction

1.1 Overview

1.2 Striated muscles

1.3 Troponin functions as a calcium sensor

1.4 Troponin I N-terminal extension

1.5 Tropomyosin regulates actomyosin interactions and stabilizes filamentous actin

1.6 C. elegans muscle

1.7 Scope and significance

2 Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationship of sarcomere components in Caenorhabditis elegans

2.1 Abstract

2.2 Introduction

2.3 Results

2.4 Discussion

2.5 Materials and Methods

2.5.1 Strains and maintenance

2.5.2 Optogenetic assays

2.5.3 Locomotion analysis

2.5.4 Statistical analysis

2.5.5 Clustering analysis

3 Molecular evolution of troponin I and a role of its N-terminal extension in nematode locomotion

3.1 Abstract

3.2 Introduction

3.3 Results and Discussion

3.3.1 Troponin evolved early in bilateria

3.3.2 Phylogenetic relationships of troponin I sequences suggest that the N-terminal extension was lost in a subset of Isoforms in the Deuterostomia

3.3.3 Troponin I N-terminal extension is required for regulation of locomotive behavior in the nematode Caenorhabditis elegans

3.4 Conclusions

3.5 Materials and Methods

3.5.1 Phylogenetic analysis

3.5.2 Nematode strains

3.5.3 Generation of transgenic nematode strains

3.5.4 Fluorescence microscopy

3.5.5 Western blot

3.5.6 Locomotion assays

3.5.7 Optogenetic assays

4 A novel alternative exon of the Caenorhabditis elegans lev-11 tropomyosin gene is used to express a head-muscle-specific isoform

4.1 Abstract

4.2 Introduction

4.3 Results

4.3.1 Identification of a novel high molecular weight tropomyosin isoform, lev-11o, containing a novel alternative Exon

4.3.2 4.3.2 Expression of Exon 7a is restricted to head and neck, while 7b is restricted to neck and main body body-wall muscles

4.3.3 lev-11 Exon 7 mutations do not change LEV-11 localization to sarcomere thin filaments

4.3.4 Levamisole resistance was isolated to regions where the mutated isoform was expressed

4.3.5 LEV-11A and LEV-11O with or without Exon 7 mutations bind to actin filaments with similar affinity

4.3.6 LEV-11O(E196K) inhibited actomyosin ATPase independent of troponin I (UNC-27) inhibitory peptide

4.4 Discussion and Conclusions

4.5 Materials and Methods

4.5.1 C. elegans strains

4.5.2 In vivo fluorescence reporter analysis of splicing patterns of lev-11 Exons

4.5.3 Levamisole sensitivity assay in C. elegans

4.5.4 Preparation of actin and LEV-11 proteins

4.5.5 F-actin co-sedimentation of LEV-11 proteins

4.5.6 Myosin ATPase activity

4.5.7 Immunofluorescence microscopy

4.5.8 Statistical analysis

5 Discussion/Conclusions

5.1 Sarcomere proteins are important in muscle

5.2 Optogenetic analysis of muscle contractility is C. elegans

5.2.1 Development of the optogenetic application in analyzing muscle kinetics

5.2.2 Sarcomeric proteins can be involved in both contraction and/or relaxation

5.2.3 Levamisole resistance correlated with the inability to maintain contraction

5.2.4 UNC-27 N-terminal extension did not affect contraction or relaxation rates in isolation

5.2.5 Future applications of optogenetics to analyze sarcomere protein function

5.3 Molecular evolution of troponin I and its N-terminal extension in nematode locomotion

5.3.1 Evolution analysis: the troponin complex appears in Bilateria

5.3.2 TNI N-terminal extension appears to be lost in a subset of Deuterostomia isoforms

5.3.3 TNI N-terminal extension within C. elegans locomotor function

5.4 C. elegans lev-11 utilizes alternative Exon 7a in a head-muscle-specific isoform: LEV-11O

5.4.1 Tropomyosin isoforms give functional variability to F-actin

5.4.2 LEV-11O is the primary isoform in the body wall muscles of the head

5.5 Model for the regulation of muscle relaxation

5.5.1 There is a currently unidentified protein (Protein X) present in the sarcomere that can modify accessibility of myosin binding sites, in a calcium-dependent manner

5.5.2a Troponin I, isoform TNI-1, may have stronger inhibitory activity than UNC-27

5.5.2b Fewer complete troponin complexes may permit greater flexibility in tropomyosin movement.

5.6 Model: lev-11 exon 7a and exon 7b may be important for regulating different thin filament qualities

5.6.1 LEV-11 (tropomyosin) Exon 7a is important for specific interactions with amino acids of TNI-3 and LEV-11 Exon 7b is important for specific interactions with amino acids of UNC-27 (TNI-3)

5.6.2a Charge differences between lev-11 Exon 7a and Exon 7b may alter interactions with troponin proteins

5.6.2b Charge differences between lev-11 Exon 7a and Exon 7b may alter binding with actin that can change LEV-11 positioning with the actin groove, to optimize favorable interactions

5.7 Future directions

5.7.1 To test our models for differential muscle kinetics in unc-27 null worms

5.7.2 To test our models for lev-11 exon 7a and 7b function in vivo

6 References

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