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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: 787-797
The Authors
Craig Mackinnon
Dr. Hiroshi Qadota
Dr. Ken Norman
•University of Illinois
•Department of Cell
Pharmacology,
Nagoya University
•University of Utah? •University B.C
•In vivo Protein
interactions in muscle
attachment structures
using FRET
•Protein interactions
in C. elegans muscle
using Y2H
•Something to do
with Zebra fish
Dr. Don Moerman
•Something to do
with muscle and
worms
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. sapiens
C. elegans
Nematode muscle
•Nematode striated muscle cells do not fuse to form a myotube
•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
Genes known to function in muscle
development
•Myosin heavy chain A-D= myo-3, unc-54, myo-2, myo-1—required for Mline 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 C29F9.7
Pat F1
98%
•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=pat4::yfp; E=pat-3::cfp;
F=overlay
Dense bodies
(arrows) & Mlines (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