Molecular determinants of the localization and assembly of the giant protein UNC-89 (obscurin) in C. elegans muscle Open Access

Xiong, Ge (2011)

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Sarcomeres, highly ordered assemblages of hundreds of proteins, perform the work of
muscle contraction. Despite increasing knowledge of sarcomere components and their
functions, we still do not have a clear understanding about how sarcomeres are assembled
and maintained during muscle contraction. Our laboratory studies sarcomere assembly in
the model genetic organism C. elegans, focusing on the function of the giant muscle
protein UNC-89. To understand the molecular mechanisms by which UNC-89 is
assembled at the M-line, and how UNC-89 performs its functions, our laboratory is
identifying its binding partners, and studying their functions.
My thesis focused on finding binding partners for two regions of UNC-89: the C-
terminal protein kinase region, and the N-terminal Ig domains 1-5. I discovered that the
PK1 protein kinase domain and interkinase region interact with LIM-9 (FHL in humans),
and that LIM-9 interacts with SCPL-1, a CTD type phosphatase, previously identified by
the lab as interacting with the kinase domains of UNC-89. I propose two structural
models for the function of these interactions at the M-line.
I have discovered a new binding partner for the N-terminal region of UNC-89, called
CPNA-1. CPNA-1 contains a conserved "copine domain" with weak homology to the
extracellular portions of integrins, and largely unknown function. I have defined a new
category of copine domain containing proteins, the "atypical" ones. CPNA-1 specific
antibodies localize to integrin adhesion complexes (M-lines and dense bodies) of
nematode body wall muscle. I found that CPNA-1 binds to the M-line proteins UNC-89,
LIM-9 (FHL), SCPL-1, UNC-96, and a protein common to the M-line and dense body,
PAT-6 (actopaxin). A genomic deletion for cpna-1, gk266, displays the typical Pat
(Paralyzed arrested at two-fold) phenotype. By localizing previously characterized
muscle adhesion complex proteins in cpna-1 mutant embryos, and localizing CPNA-1 in
other Pat mutants, I have placed CPNA-1 in the M-line/dense body assembly pathway of
embryonic muscle. By using RNAi and mutants, I have begun to define a role for CPNA-
1 in adult muscle. I conclude with a model that PAT-6 recruits CPNA-1, and in turn,
CPNA-1 recruits additional proteins (UNC-89, LIM-9, SCPL-1, UNC-96) to the M-line.

Table of Contents

Table of Contents

1. Chapter 1: Introduction 1.1 Muscle Organization 1.2 Use of C. elegans to Study Muscle Sarcomere Organization and Assembly 1.3 Giant Polypeptides in Muscle 1.3.1 C.elegans Giants 1.3.2 Human Titin 1.3.3 Obscurin (Human UNC-89) 1.3.4 UNC-89 2. Chapter 2: A LIM-9 (FHL) / SCPL-1 (SCP) Complex Interacts with the C-terminal Protein Kinase Regions of UNC-89 (Obscurin) in C. elegans Muscle 2.1 Introduction 2.2 Results 2.3 Discussion 2.4 Materials and Methods 3. Chapter 3: CPNA-1, A Novel Copine Containing Protein, Links Integrin Associated Protein PAT-6 (Actopaxin) to the Giant Protein UNC-89 (Obscurin) at Muscle Adhesion Site in C. elegans 3.1 Introduction 3.2 Results 3.3 Discussion 3.4 Materials and Methods 4. Chapter 4: Summary and Future Directions 4.1 CPNA-1 4.2 A LIM-9/SCPL-1 Complex Interacts with the C-terminal Portion of UNC-89 5. References

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