Muscle Cell Biology and Development

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Muscle Cell Biology and Development "Sarcomere
Assembly"
Aikaterini Kontrogianni-Konstantopoulos,
Ph.D. Dept. of Physiology Office
number HSF1, 580 D e-mail
akons001_at_umaryland.edu
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Outline
Introduction to Myofibrillogenesis Current
Models for Sarcomere Formation Giant Muscle
Proteins "Molecular Rulers" for Myofibril
Assembly
Titin
Nebulin
Obscurin
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Myofibrillogenesis
Myofibrillogenesis is a highly complex process
that requires the ordered integration of actin,
myosin and hundreds of proteins into their
functional unit, the sarcomere.
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Sarcomere Assembly Two Prominent Models

• I. Premyofibril Model (J.W. Sanger and
  colleagues)
• II. Independent Assembly of Thin and Thick
  Filaments
• (H. Holtzer and colleagues)

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The Premyofibril Model Main Points
(J.W. Sanger and colleagues)
Three distinct structures are formed during
myocyte development premyofibrils, nascent
myofibrils and mature myofibrils.
Premyofibrils contain transitory arrays of I-Z-I
complexes, consisting of sarcomeric actin
occupying primitive I-bands, attached to
precursors of Z-disks, termed "Z-bodies",
enriched in a-actinin, that interact with
miniature A-bands, composed of non-muscle
myosin II. Premyofibrils progress to nascent
myofibrils, which develop into mature
myofibrils with the concurrent replacement of
non-muscle myosin II by muscle myosin II.
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The Premyofibril Model
(J.W. Sanger and colleagues)
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The Premyofibril Model Key Feature
Precursors of thin and thick filaments form along
the SAME STRUCTURES, which together develop into
mature sarcomeres.
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The "Independent Assembly" Model Main Points
(H. Holtzer and colleagues)
Microfilament bundles resembling the stress
fibers of non-muscle cells act as a scaffold
during sarcomere assembly. I-Z-I complexes
containing ?-actinin, sarcomeric actin and the
NH2- terminus of titin are organized in
register on these filamentous structures to
form non-striated myofibrils in the absence of
muscle myosin containing thick filaments.
Full-length thick filaments assemble and
incorporate independently into these preformed
structures.
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The Premyofibril Model Key Feature
Precursors of thin and thick filaments form along
the SAME STRUCTURES, which together develop into
mature sarcomeres.
The Independent Assembly Model Key Feature
The earliest precursors of mature thin and thick
filaments FORM INDEPENDENTLY in the myoplasm of
developing muscle, but later stages of
development proceed along common filaments.
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What Both Models Agree On?
Obscurin
Myomesin
Structural proteins are essential to the proper
assembly and incorporation of actin and myosin
into mature myofibrils. During the initial
assembly of myofibrils, Z-bodies composed of
a-actinin, the NH2-terminal region of titin,
nebulin, and T-cap nucleate the polarized
organization of thin actin filaments to form
"I-Z-I brushes" that become incorporated into
forming sarcomeres. Likewise, proteins of the
M-line, including the COOH-terminal region of
titin, myomesin, obscurin and M-protein play a
key role in the integration of myosin thick
filaments into periodic A-bands.
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Muscle Giants Molecular Templates in
Sarcomerogenesis
Three giant, muscle-specific proteins, appear to
govern the regular organization of
sarcomeres I. Titin (3-4 MDa)
II. Nebulin (800 kDa) and
III. Obscurin (800 kDa)
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The Modular Titin (3-4 MDa)
Titin is the third most abundant muscle
protein. It is 1.6 ?m in length a single
titin molecule spans half the sarcomere,
anchoring its NH2- and COOH-termini in the Z-disk
and M-line, respectively. Titin is modular in
structure 90 of its mass consists of
repeating immunoglobulin-C2 (Ig-C2) and
fibronectin-III (Fn-III) domains that provide
binding sites for diverse myofibrillar proteins.
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The Modular Titin (3-4 MDa)cont.
The remaining 10 of its mass consists of
unique non-repetitive sequence motifs,
including phosphorylation sites, binding sites
for muscle specific calpain proteases and
COOH-terminal Ser/Thr kinase domains. The
C-terminal 2 MDa of titin are located within the
A-band, where titin tightly associates with
the myosin thick filaments and several A-band
proteins such as MyBP-C, myomesin and
M-protein. The most C-terminal end of the
molecule (200 kDa), which is embedded in the
M-line, contains a Ser/Thr kinase domain, which
implicates titin in myofibrillar signal
transduction pathways. In the I-band, titin
(800 kDa to 1.5 MDa) carries Proline/Glutamate/V
aline/ Lysine (PEVK) rich sequences, which
confer great extensibility to the molecule.
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The Modular Titin (3-4 MDa)cont.
At the junction of the I-band with the Z-disk,
titin interacts with the actin filaments. The
N-terminal 80-kDa region of titin spans the
entire Z-disk. Several copies of a 45-residue
repeat, the Z-repeat, bind ?-actinin within the
Z-disk. The two most N-terminal Ig-domains of
titin that reside in the periphery of the
Z-disk bind another Z-disk protein, T-cap, and a
small isoform of ankyrin 1, sAnk1, that is
selectively enriched in the sarcoplasmic
reticulum membranes.
Two major functions have been attributed to
titin in striated muscle I. molecular
blueprint that specifies and coordinates the
precise assembly of many of the
structural, regulatory and contractile proteins
that compose the sarcomere, and II.
molecular spring that gives striated muscle its
distinct biomechanical properties and
integrity during contraction, relaxation and
stretch .
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The Nebulous Nebulin (800 kDa)
Nebulin is a giant actin-binding protein of
vertebrate striated muscle. In situ, a
single molecule is incorporated into and is
co-extensive with the thin filaments of
a-actin, that form the I-band and interact with
myosin to produce contraction. Its
NH2-terminus extends to the pointed ends of thin
filaments, whereas its COOH-terminus is
partially inserted into the Z-disks.
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The Nebulous Nebulin (800 kDa)cont.
Most of its mass (97) is composed of 185
modular, 35-amino acid repeats. The central
154 modules are organized into 22 super-repeats
of 7 modules each, which may complement the
periodicity of the actin filaments. Nebulin
isoforms of different sizes, generated by
alternative splicing of a single transcript,
correspond to the various sizes of thin filaments
present in developing and adult muscle
fibers. The extreme COOH-terminal end of
nebulin contains a Ser-rich region with
multiple phosphorylation sites and an SH3 domain
that binds to myopalladin, a Z-disk protein.
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The Nebulous Nebulin (800 kDa)cont.
In addition to its lateral interactions with
actin, nebulin contains distinct sites at its
NH2-terminus that interact with two
actin-associated proteins, tropomyosin and
troponin I/C/T, and the thin filament capping
protein, tropomodulin, providing a mechanism
for terminating the growth of the actin filaments
at precisely the length of nebulin. Thus,
nebulin is the prime candidate molecule for
functioning as a "ruler"to specify the
precise lengths of the actin thin filaments in
skeletal muscle.
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Titin and Nebulin Molecular Blueprints for
Sarcomere Assembly
Between titin and nebulin, two molecular
templates are available that associate with, and
presumably help to organize, the LONGITUDINAL
DIMENSIONS of the forming sarcomere.
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Obscurin a Multitasking Muscle Giant
Obscurin is the third giant protein of the
contractile apparatus identified in
vertebrate striated muscle. Like its
predecessors, it is a multi-domain protein
composed of adhesion modules and signaling
domains arranged mostly in tandem. Its
NH2-terminus contains 54 Ig-C2 and 2 FnIII
domains, followed by an IQ motif and a
conserved SH3 domain adjacent to Rho-guanine
nucleotide exchange factor (Rho-GEF) and
pleckstrin homology (PH) domains. The
COOH-terminal end of the protein consists of 2
additional Ig domains followed by a
non-modular region of 420 amino acid residues
that contains several copies of a consensus
phosphorylation motif for ERK kinases.
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Obscurin a Multitasking Muscle Giantcont.
The obscurin gene, obscurin-MLCK, also encodes
two ser/thr kinase domains, but these are
apparently not expressed as part of the 800 kDa
form of the protein, and instead are made as
smaller, alternatively spliced products, mainly
in heart. Unlike titin and nebulin, which are
integral components of sarcomeres, obscurin is
not present within sarcomeres but intimately
surrounds them, primarily at the level of the
Z-disk and M-line, where it is appropriately
positioned to participate in their assembly
and integration with other sarcoplasmic elements.

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Intracellular Localization of Obscurin and Titin
in Adult Skeletal Muscle
Obscurin Titin-Z
C
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Obscurin a Multitasking Muscle Giantcont
Obscurin interacts with diverse protein
partners located in distinct compartments
within the cell, including sAnk1, an integral
component of the sarcoplasmic reticulum
membranes, as well as sarcomeric myosin and
MyBP-C. The unique localization of obscurin
around the periphery of the myofibrillar
Z-disk and M-line further suggests that it may
specify the PERIMETER of the sarcomere.
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TAKE-HOME MESSAGE!!!
The unique structural properties and
subcellular locations of titin, nebulin and
obscurin suggest that they are responsible for
organizing the LONDITUDINAL DIMENSIONS of
sarcomeres into thin and thick filaments,
specifying the PERIMETER of developing sarcomeres
and coordinating the SARCOMERIC ALIGNMENT of
nearby structures, like the sarcoplasmic
reticulum. Consistent with this, all three
giant proteins have been linked, either directly
or indirectly, to several forms of
cardiomyopathies and muscular dystrophies,
demonstrating their critical role in normal
muscle development and physiology.
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Selected Reading

• Sanger JW et al., Clin. Orthop, S153-162, 2002
• Schultheiss T et al., J Cell Biol, 1101159-72,
  1990
• Gregorio CC et al., Curr Opin Cell Biol 11
  18-25, 1999
• Wang K, Adv Biophys 33 123-134, 1996
• McElhinny AS et al., Trends Cardiovasc Med 13
  195-201, 2003
• Kontrogianni-Konstantopoulos A et al., Mol Biol
  Cell 14 1138-1148, 2003
• Gautel M et al., Rev Physiol Biochem Pharmacol
  138 97-137, 1999
• Clark KA et al., Annu Rev Cell Dev Biol 18
  637-706, 2002