Molecular genetic analysis of unc-94 and unc-100, two genes that are important for normal myofibril organization in C. elegans Público
Wortham, Tesheka (2010)
Abstract
Sarcomeres, highly ordered assemblages of several hundred proteins, perform the work of muscle contraction. Despite increasing knowledge of sarcomeric proteins and their functions, we lack a clear understanding about how sarcomeres are assembled and maintained. We study this question in the model organism, C. elegans. My studies focused on phenotypically characterizing and molecularly identifying two genes that are important for normal sarcomere assembly and maintenance: unc-94 and unc-100. Along with the original allele of unc-94, su177, I have recovered and characterized a new allele, sf20. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some, abnormally accumulated near muscle cell-to-cell boundaries. EM shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. I have proven that unc-94 encodes a tropomodulin, a conserved protein known to cap the pointed ends of thin filaments. su177 is a splice site mutation specific to isoform unc-94a; sf20, has a stop codon, which is shared by both unc-94a and unc-94b. Promoter-GFP analysis revealed that unc-94a isexpressed mainly in body wall muscle, whereas unc-94b is expressed mainly in pharyngeal muscle. Anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). These antibodies localize UNC-94 to two closely spaced parallel lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell to cell boundaries. For unc-100, I characterized two alleles: su115 and su149. By polarized light, myofibrils of unc-100 animals are disorganized. EM shows missing M-lines, broken dense bodies, and a lack of defined A and I-bands. Staining of su115 muscle with antibodies to a variety of known sarcomeric proteins revealed disruption of thick filaments, M-lines and dense bodies. We used three-factor and SNP mapping to place unc-100 within a 500 kb region. After performing transgenic rescue, RNAi and/or sequencing many candidate genes within the given region, we have yet to determine the molecular identity of unc-100.
Table of Contents
Chapter 1: Introduction……………………………………………………………….1
Part 1: Purpose……………………………………………………………2
Part 2: Muscle Structure and Function………………………………....3
Part 3: C. elegans as a Model for Studying Muscle…………………....12
Part 4: Actin Filament Assembly……………………………………….21
Part 5: The Roles of Actin Regulatory Proteins in Myofibril
Assembly and Maintenance………………………………….....25
Part 6: Muscle Focal Adhesions………………………………………...32
hapter 2: unc-94 Encodes a Tropomodulin in Caenorhabditis elegans…………45
Introduction……………………………………………………………...46
Results……………………………………………………………………50
Figures…………………………………………………………………...58
Discussion………………………………………………………………..72
Materials and Methods………………………………………………….78
Chapter 3: Genetic and Molecular Characterization of unc-100, a Gene Required
for Normal Myofibril Maintenance or Growth, but not Initial
Assembly, in C. elegans…………………………………………………86
Introduction……………………………………………………………...87
Results and Discussion………………………………………………….88
Figures…………………………………………………………………...97
Materials and Methods………………………………………………..114
Chapter 4: Conclusions and Future Directions…………………………………..120
Literature Cited…………………………………………………………………….127
LIST OF TABLES
Table 3.1 Candidate gene mapping of unc-100.……………………………104
Table 3.2 SNP mapping data for dpy-5 unc-100 and unc-29 unc-100
recombinants……………………………………………………...106
Table 3.3 Individual SNP data………………………………………………107
Table 3.4 unc-15 and dcp-66 sequence comparison………………………..112
Table 3.5 RNAi (by feeding) of predicted genes on cosmids in set A
(candidate gene mapping of unc-100)……………………………113
LIST OF FIGURES
Figure 1.1 Structure of the skeletal muscle sarcomere………………………..8
Figure 1.2 The myosin head uses ATP to pull on an actin filament………….9
Figure 1.3 T tubules and the sarcoplasmic reticulum………………………..11
Figure1.4 The body-wall muscle of C. elegans………………………………17
Figure 1.5 Polarized light images of wt, unc-89 and alleles of unc-96……...19
Figure 1.6 Embryogenesis in wild-type and Pat mutants…………………...20
Figure 1.7 Polymerization of G-actin in vitro occurs in three phases……...24
Figure 1.8 Troponin and tropomyosin interact to control the attachment
of crossbridges to actin…………………………………………....30
Figure 1.9 Organization of accessory proteins in a sarcomere……………..31
Figure1.10 Integrins mediate linkage between fibronectin in the
extracellular matrix and the cytoskeleton……………………….40
Figure1.11 The costameric cytoskeleton of striated muscle………………....42
Figure 1.12 Protein-protein interactions at M-lines and dense bodies…….. 43
Figure 2.1 unc-94 mutants show disorganized muscle structure,
decreased motility and low brood size………………………….58
Figure 2.2 Immunofluorescent localization of several known
sarcomeric proteins in wild type and unc-94 mutant muscle..60
Figure 2.3 Electron micrographs of body-wall muscle from wild type
and from unc-94(sf20) …………………………………………..61
Figure 2.4 Figure 2.4 Genetic and physical mapping of unc-94,
location of mutation sites for su177 and sf20, and the
sequences of UNC-94a and b…………………………………...62
Figure 2.5 Fluorescent images of GFP expression in transgenic
animals that carry unc-94 promoter elements………………..64
Figure 2.6 By Northern blot, unc-94 mutations result in decreased
levels of unc-94 mRNAs………………………………………..65
Figure 2.7 By Western blotting, UNC-94 polypeptides can be detected
from wild-type, but are absent from or in reduced amounts
in unc-94 mutants or RNAi animals…………………………..66
Figure 2.8 By immunofluorescence, UNC-94 localizes to the pointed
ends of thin filaments and to muscle cell boundaries………..67
Figure 2.1S Electron micrographs of body-wall muscle from wild type
and from unc-94(sf20) ……………………………………….…69
Figure 2.2S Comparison of tropomodulin sequences from worms,
flies and humans…………………………………………….…70
Figure 3.1 Polarized light microscopy of body-wall muscle from
unc-100 adults……………………………………………….…97
Figure 3.2 Liquid motility and brood size assays for unc-100 animals...99
Figure 3.3 Immunofluorescent localization of several known sarcomeric
proteins in wild type and unc-100 adult body-wall
muscle ………………………………………………………....100
Figure 3.4 Electron micrographs of adult body-wall muscle from wild
type and the two unc-100 mutants ……………………….….101
Figure 3.5 Immunofluorescent localization of MHC A in wild type
and unc-100 (su149) embryos……………………………......102
Figure 3.6 Deficiency mapping of unc-100 ……………………………..103
Figure 3.7 Mapping of unc-100 …………………………………………105
Figure 3.8 Nomarski images of the gonad of su115 and su149 adult
animals……………………………………………………….108
Figure 3.9 Transgenic Rescue of unc-100……………………………...109
Figure 3.10Predicted genes on cosmid C26C6………………………….110
Figure 3.11 Deficiency mapping of unc-100……………………………..111
Figure 4.1 A model of synergistic regulation of sarcomeric actin
organization by Tmod, ADF/cofilin, AIP1, and profilin….126
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