The PIX-1 Signaling Pathway that Includes the Rho GEF PIX-1 and the Rho GAP RRC-1 Direct Assembly and Stability of Integrin Adhesion Complexes in Striated Muscle Open Access
Moody, Jasmine (Spring 2022)
Abstract
Integrin adhesion complexes (IACs) permit the attachment of cells to the extracellular matrix (ECM) and are essential for tissue and organ development and cell motility. Although much is known about the composition and initial assembly of IACs, what determines where and when an IAC will form remain unknown. In striated muscle, IACs attach the muscle cell to the ECM and transmit the force of muscle contraction. C. elegans is an excellent model to study muscle assembly, maintenance, and regulation and their striated muscle has IACs at the M-line, dense body, and attachment plaque at muscle cell boundaries (MCBs). I discovered that loss of function of the gene pix-1 results in the absence of IAC components only at MCBs. PIX-1 deficiency or overexpression results in disrupted MCBs, decreased whole animal locomotion, and muscle-specific expression of wildtype PIX-1 rescues the MCB phenotype. PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42 and have numerous functions in various cell types. Mutations in genes encoding proteins at known steps of the PIX signaling pathway, including the Rac, CED-10, the scaffold GIT-1, and the effector protein kinases, PAK-1, and PAK-2, show defects at MCBs. Either constitutively active or catalytically dead protein kinase pak-1 mutants also show disrupted MCBs, indicating that increased or decreased output of the PIX pathway yields the same phenotype. Rho GTPases function as molecular switches between active and inactive states, via GEF and GAP (GTPase activating) proteins respectively. However, a GAP for the PIX pathway had not been identified for any organism or cell type. Upon screening mutants of 18 muscle-expressed genes that encode proteins with RhoGAP domains, I discovered that RRC-1 deficiency yields the MCB defect. rrc-1 loss of function mutants lack IAC protein accumulation at MCBs and have reduced motility. RRC-1 and PIX-1 both localize to MCBs and show complex genetic interactions. Together this is the first evidence of a role for the PIX pathway in muscle and the discovery of a GAP for the pathway. An important goal for future research is to understand the molecular mechanisms by which the PIX pathway affects the assembly of IACs.
Table of Contents
Chapter 1: Introduction…………………………………………………………………...........1
Part I: Purpose……………………………………………………………………...2
Part II: Muscle structure and function……………………………………………5
Part III: C. elegans as a model for gaining new insights into muscle assembly, maintenance, and regulation....12
Part IV: Rho-family GTPase Signaling and Roles in Cytoskeleton dynamics….................................18
Part V: Integrin adhesion complexes in muscle ………………………………….....................................35
Chapter 2: The Rho-GEF PIX-1 Directs Assembly or Stability of Lateral Attachments Structures between Muscle Cells
Introduction…………………………………………………………………..…….44
Results………………………………………………………………………………47
Figures……………………………………………………………………………....56
Discussion…………………………………………………………………………..82
Material and Methods……………………………………………………………..87
Chapter 3: The RhoGAP RRC-1 is Member of the PIX Pathway and Controls Assembly or Stability of Integrin Adhesion Complexes in Muscle
Introduction……………………………………………………………………….100
Results…………………………………………………………………………….104
Figures…………………………………………………………………………….111
Discussion………………………………………………………………………...133
Materials and Methods………………………………………………………….137
Chapter 4: Conclusion and Future Directions……………………………………………...143
Chapter 5: Appendix/Miscellaneous Data………………………………………………….151
Works Cited…………………………………………………………….....……………………..160
LIST OF TABLES
Table 2.1 DH domain proteins in C. elegans muscle………………………………….…81
Table 3.1S RhoGAP Screening Results in tabular form………………………………...129
Table 5.1 Sequence Analysis of MMP hum-7 mutant alleles………………………….156
LIST OF FIGURES
Figure 1.1 Organization of the contractile apparatus in vertebrate skeletal muscle….............................10
Figure 1.2 C. elegans are an excellent model to study striated muscle structure and function……………….16
Figure 1.3 GEF & GAP proteins are two main classes regulators of GTPase activity..................................32
Figure 1.4 GDIs are the third and less characterized class of regulators that facilitate GTPase activity…....33
Figure 1.5 Integrin adhesion complexes (IACs) are assembled and disassembled via a dynamic, stepwise mechanism in motile cells……………...............................................................................…………………....39
Figure 1.6 Costameres are IACs in vertebrate striated muscle (skeletal and heart muscle)……………………40
Figure 1.7 Integrin adhesion cells are localized at M-lines, dense bodies, and muscle cell boundaries in C. elegans body wall muscle........................................................................................................................41
Figure 2.1 Identification of pix-1 as a gene required for the assembly of the IACs at muscle cell boundaries….56
Figure 2.2 By N-SIM confocal microscopy, Live imaging of cortical F-actin at muscle cell boundaries………58
Figure 2.3 PIX-1 is most similar to b-PIX and, by Western blot analysis, acts as a GEF for CED-10 (Rac) in muscle……………………………..................................................................................................................…...60
Figure 2.4 Both loss of function and overexpression of pix-1 results in reduced locomotion………………………62
Figure 2.5 By western blot, Overexpression of pix-1 results in the disruption and CRISPR repair of pix-1(gk299374) results in normalization of muscle cell boundary…………………………………..................……………..64
Figure 2.6 Antibodies to PIX-1 detect PIX-1a on a western blot and localize to muscle cell boundaries, M-lines, and dense bodies……………................................................................................................……………………...66
Figure 2.7 Mutations in genes encoding known proteins of a PIX-1 pathway result in muscle cell boundary disruption……………………….............................................................................................……………………....68
Figure 2.8 PIX-1 levels are reduced in git-1 and pak-1 mutants………………......................................…………69
Figure 2.9 P190 is conserved in PIX RhoGEF domains and required for PIX-1 function at the muscle cell boundary………………….........................................................................................………………………………….70
Figure 2.10 P190S may alter RhoGEF structure and interaction with Rac, and in muscle there is a reduction in activated Rac………………………………………….................................................................................................72
Figure 2.1S Immunostaining of 6 pix-1 mutant alleles using antibodies to PAT-6………74
Figure 2.2S Live imaging of cortical F-actin at muscle cell boundaries…………………..75
Figure 2.3S A pix-1 mutant has normally organized sarcomeres…………………………76
Figure 2.4S PAT-6 immunostaining of two additional alleles of rac-2 and ced-10………77
Figure 2.5S 3D rendering of SIM images of PAT-6 muscle boundary localization of wildtype compared with pix-1(gk893650)………………………………...................................................................……..78
Figure 2.6S Root mean square fluctuation analysis (RMSF) of PIX-1-Rac complexes indicate that the P190S mutation alters stability at the interface……….............................................................79
Figure 2.7S Yeast two-hybrid assays show that full-length PAK-1 interacts specifically with full-length PIX-1a but not full-length PIX-1b or full-length GIT-1…...80
Figure 3.1 Either catalytically dead or constitutively active PAK-1 kinase results in a muscle cell boundary defect…………………………………………………..111
Figure 3.2 Loss of function rrc-1 mutants show lack of or disorganization of PAT-6 at the muscle cell boundaries…………………………………………………….112
Figure 3.3 Additional IAC components show disruption at the muscle cell boundaries in rrc-1 mutants…………………………………………………………………..113
Figure 3.4 Loss of function rrc-1 mutants show reduced whole animal locomotion….114
Figure 3.5 HA-tagged RRC-1 localizes to muscle cell boundaries………………….….115
Figure 3.6 PIX-1 is required for the proper localization of RRC-1 but not the stability of RRC-1………….116
Figure 3.7 RRC-1 is not required for the localization or stability of PIX-1……...……118
Figure 3.8 GIT-1 is not required for the localization of RRC-1, but GIT-1 is required for the stability of RRC-1……………………………………………….......................................................…….120
Figure 3.9 Double mutant analysis of rrc-1 and pix-1 show genetic interaction………122
Figure 3.10 RRC-1 is RhoGAP in the PIX-1 pathway…………………………………....124
Figure 3.1S RhoGAP Screening Results…………………………………………………...125
Figure 3.2S Sequence Alignment of RRC-1 and human ARHGAP33…………………..129
Figure 3.3S sgRNAs and repair template information for generation of CRISPR/Cas9 strains.......131
Figure 4.1 The PIX Pathway has a role in muscle………………………………………150
Figure 5.1 HUM-7 loss of function causes disruption of PAT-6 localization only at the attachment plaques between muscle cells………………………………….............................................…155
Figure 5.2 unc-73 loss of function alleles exhibits either disruption or loss of PAT-6 at muscle cell boundaries…………………………………………………...................................……...158
Figure 5.3 Cardiomyocyte cross-sectional areas are significantly increased in b-PIX conditional knockout…………………………………………….....................................…………….159
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