C. elegans PAT-4/ILK Functions as an Adaptor Protein within Integrin Adhesion Complexes Mackinnon, C., Qadota, H., Norman, K., Moerman, D., Williams, B. (2002) Current Biology 12:

Craig Mackinnon University of Illinois In vivo Protein interactions in muscle attachment structures using FRET Dr. Hiroshi Qadota Department of Cell Pharmacology, Nagoya University Protein interactions in C. elegans muscle using Y2H Dr. Ken Norman University of Utah? Something to do with Zebra fish Dr. Don Moerman University B.C Something to do with muscle and worms The Authors

Sarcomere assembly and muscle attachment in C. elegans. Summary: Muscle sarcomere assembly is a highly orchestrated affair involving several proteins. The steps involved in initiating the correct placement of sarcomere substructures are poorly understood. Using mutants in C.elegans we are attempting to dissect the various steps in this process. The model of sarcomere assembly is based on observations that suggest assembly initiates at the plasma membrane and involves proteins within muscle and within the extracellular matrix. This process is remarkably similar to adhesion complex assembly in non muscle cells.

Why use C. elegans as a model system to study muscle development and sarcomere assembly?

To understand muscle development and muscle contraction in humans

The Sarcomere H. sapiensC. elegans

Nematode muscle Adhere tightly to adjacent muscle cells in a quadrant Myofilament lattice anchored to membrane via lateral attachments Adult muscle cell 10 A-bands wide Thin filaments anchor thick filaments to Dense bodies Obliquely striated..not cross striated Nematode striated muscle cells do not fuse to form a myotube

Genes known to function in muscle development Myosin heavy chain A-D= myo-3, unc-54, myo-2, myo-1—required for M- line assembly Mysin head= unc-54—required for filament assembly Myosin light chains: mlc-1, mlc-2, mlc-3—required for calcium of myosin ATPase Paramyosin= unc-15—required for organized body wall muscle; proper assembly of myosin? Twitchin Protein= unc-22—required for proper organization of sarcomeres; regulation of muscle contraction

Genes known to function in muscle development M-line proteins: UNC-89, creatine kinase, myomesin, skelemin, M protein, titin—required for proper maintenance of the M-line and for holding thick filaments in register Actin filaments: act-5, unc-92, act-1, act-3—required for cell shape; cell movement; muscle contraction Troponin-Tropomyosin: lev-11, pat-10, mup-2—required for viabilty; proper contraction/relaxation Thin filament associated: unc-60, unc-87—required for thin filament assembly

Genes required for early sarcomere organization Mup class (muscle positioning) and Pat class (paralyzed and arrested elongation at twofold) Dense body & M-line: Deb-1/vinculin; pat-3/ß-integrin, pat-2/α-integrin, unc-52/perlecan Pat class: unc-112, unc-52, myo-3, lev-11

Sarcomere assembly & membrane Linkage between myofibril and membrane provided by Dense body --Major constituents of Dense body: α-actinin, vinculin, talin --anchor of myofilament lattice: α, β-1 integrin components --extracellular anchor: perlecan (unc-52) *Pat mutants studies to date: unc-52, pat-3, unc-112, unc-97, deb-1 This paper focuses on pat-4

Pat-4 is a homolog of vertebrate ILK RNAi on C29F9.798% Pat F1 Genomic fragment C29F9.7 rescues pat-4 phenotype a. Localized to LGIII b. Genomic structure of pat-4 (exons=gray) c. pat-4 point mutations st551, st579 d. domain analysis

PAT-4 and βPAT3 Integrin Colocalize at Muscle Attachments A.pat-4::gfp localizes to dense bodies B.Abs to βPAT3 integrin C.Overlay of A, B D.Detail of body wall muscle (D-F). D=pat- 4::yfp; E=pat-3::cfp; F=overlay Dense bodies (arrows) & M- lines (arrow head)

PAT-4/ILK required for Dense body & M-line assembly Embryos N2 & pat-4 430min post-fertilization hypodermis-hypodermis junction muscle quadrant A, B. Normal C, D. some irregularity; some foci not in tight stripe of attachments E, F. vinculin does not assemble properly into nascent attachments G, H. UNC-89, an M-line protein, fails to polarize and remains in clumps in the cytoplasm

PAT-4/ILK required for UNC-112 assembly into nascent attachments A.430 min. post-fertilization, ßPAT3 integrin located in nascent muscle attachments B.UNC-112::GFP observed in some foci (arrow), but mostly does not colocalize with integrin

Unc-52, pat-2, unc-112 required for PAT-4/ILK assembly -Embryo’s stained with Abs to PAT-4/ILK -Perlecan, Integrin, UNC-112 required for polarization -Vinculin not required

Y2H Screen: UNC-112 interacts with PAT-4/ILK

PAT-4/ILK acts as an adaptor during attachment assembly ILK functions upstream of vinculin & UNC-89 and downstream of perlecan & integrin Essential function of PAT-4/ILK independent of kinase activity (results not shown)…thus acts primarily as an adaptor

Summary Pat-4 encodes the sole ILK homolog in C. elegans PAT-4/ILK acts as an adaptor to link proteins at the adhesion complex Absence of PAT-4/ILK blocks sarcomere assembly Y2H identified UNC-112 as an interacting partner of PAT-4/ILK ILK kinase domain is vestigial Proper PAT-4/ILK localization requies perlecan & integrin, but not vinculin