MYOGENIC STAGE, SARCOMERE LENGTH AND PROTEASEACTIVITY MODULATE LOCALIZATION OF MUSCLE-SPECIFICCALPAIN*
Koichi Ojima,1,2 Yasuko Ono,1 Naoko Doi,1,2 Katsuhide Yoshioka,3 Yukiko Kawabata,2,3
Siegfried Labeit,4 and Hiroyuki Sorimachi1,2
From 1Department of Enzymatic Regulation for Cell Function, The Tokyo Metropolitan
Institute of Medical Science, Tokyo 113-8613, Japan
2
3

CREST, JST, Kawaguchi 332-0012, Japan

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657,
Japan

4

Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim,
Mannheim 68167, Germany
Running title: Protease activity-dependent myofibrillar localization of muscle-specific calpain

Address correspondence to: Hiroyuki Sorimachi, Department of Enzymatic Regulation for
Cell Function, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan,
Tel: +81-3-3823-2181; Fax: +81-3-3823-2359; E-mail: sorimach@rinshoken.or.jp
p94/calpain3
intracellularisaprotease

Ca2+-binding

as the sarcomere lengthens beyond ca. 2.6
andpredominantly

μm

2.8

for

wild-type

andexpressed in skeletal muscles. p94 binds toprotease-inactive p94, respectively. Thesethe N2A and M-line regions of connectin/titin,

data demonstrate for the first time that p94

andGenetic

proteolytic activity is involved in responses toevidence showing that compromised p94

muscle conditions, which may explain why

proteolytic

activity

p94 inactivation causes LGMD.

dystrophy(LGMD-type)

localizes

intheZ-bands.

leads

to

muscularindicates

theimportance of p94 function in myofibrils.

p94/calpain 3 is a member of theHere, we show that a series of p94 splicecalpain family of intracellular Ca2+ requiring

variants are expressed immediately after

Cys proteases, which cleave substrates atmuscledifferentially

specific and limited sites to modulate theirchange localization during myofibrillogenesis.

structure and function. In contrast to the

We found that endogenous N-terminal (butconventional μ- and m-calpains, which are

not C-terminal) domain of p94 were not only

ubiquitously expressed in almost all cells (1,2),

localized in the Z-bands but also directly

p94 is predominantly expressed in skeletalbound to sarcomeric α-actinin. These data

musclesuggest the incorporation of proteolytic

differentially spliced variants in skeletal muscle

N-terminal fragments of p94 into the Z-bands.

and nonmuscle cells (3). The human p94 gene,

In myofibrils, localization of exogenously

CAPN3, was identified as responsible forexpressed p94 shifted from the M-line to N2A

limb-girdle

differentiation

and

1

with

lessermuscularamounts

dystrophyofseveral

type

2A

(LGMD2A, or “calpainopathy”) (4). In mice,(15,16), T-cap/telethonin (17), muscle-specifictransgenic overexpression of a protease-inactive

RING-finger protein-1 (MURF-1) (18), as wellform of p94, p94:C129S, and p94 geneas p94 (19). These proteins interact withknock-out

causedandconnectin at three significant regions in themusculardystrophy,(5-7).

sarcomeres, namely, Z-bands, N2A and M-line

a

mild

myopathy

respectively

Furthermore, a defect in the proper proteolytic

regions,

activity of p94 is a common feature ofcomplexes form. p94 directly interacts with

LGMD2A pathogenic mutations (8). These

connectin at both N2A and M (19). Interaction

findings indicate that p94 protease activity is

between p94 and connectin at N2A stabilizes

essential to maintain healthy condition of

p94, which otherwise autolyzes very rapidly,

skeletal

specific

thus, connectin may regulate p94 proteolytic

physiological functions of p94 as a protease infunctions (20,21). p94 has been also detected inskeletal muscle remain largely unknown.

Z (19,22), although a molecular basis formuscle.

However,

the

where

highlyorganized

proteinhighlyanchoring p94 in Z is unknown. Thus, p94 exists

organized sarcomere structures, which consist of

in all three regions in sarcomeres, suggesting

systematically expressed myofibrillar proteins.

that

One of the earliest myofibrillar proteinsorganization in cooperation with other connectin

expressed in vertebrate myogenic cells isligands and/or connectin.

Skeletalmuscles

containp94

maycontributetomyofibrilconnectin/titin (9), which is the largest protein

This study aims (1) to characterizemolecule known, and is abundantly expressed in

the spatiotemporal expression and localization

striatedanof p94 and its splicing variants during skeletal

Duringmuscle differentiation in relation to connectin,

sarcomere assembly, connectin is considered a(2) to identify p94 interacting molecules in the

key

thin

Z-band components, and (3) to clarify the rolefilament/Z-band precursors (I–Z–I bodies) andof the proteolytic activity of p94 in the unique

the thick filaments, which are independently

context

assembled

immunohistochemical

muscles

intrasarcomericmolecule

in

where

it

filament(10,11).

forgrowthconstitutes

integration

tips

of

of

elongatingof

musclestudy

cells.
using

Detailed
primary

myotubes. The N-terminus of connectin iscultures of skeletal muscle cells revealed thatlocated in both I–Z–I body and mature Z, and

p94 and its splice variants differ in their

the C-terminus is in the M-line of the thickexpression and localization during maturation offilament (12-14). A single molecule of connectinsarcomere structures. It was also demonstrated

spans a half of the sarcomere from Z to M tothat correlation between localization of p94 andintegrate the Z-band and the thick filaments,

sarcomere lengths exists, which, at least in part,

maintaining the location of thick filaments

involves its protease activity.

between the Z-bands.
Experimental procedures

Connectin also plays an importantrole as a scaffold for its specific ligands such as

Experimental animals - All procedures usedsarcomeric-α-actinin/α-actinin 2 (s-α-actinin)2

for experimental animals were approved bysoybean trypsin inhibitor and 2 mM PMSF).

the Experimental Animal Care and Use

The amount of protein was quantified with aCommittee

DC

in

the

Tokyo

Metropolitan

RC

protein

assay

kit

(Bio-Rad

Institute of Medical Science. C57BL/6 mice

Laboratories Inc.). Equal amounts of protein

were purchased from Nihon CLEA Inc.

from

Preparation of mouse skeletal muscle cell -SDS-PAGE. Gels were transferred onto

Mouse skeletal muscle cells were prepared as

Immobilon-P transfer membrane (Millipore

previously described (23). Isolated myogenic

Corp.). After blocking, membranes were

cells

incubated

wereculturedon

Matrigel

(BD

each

samplewith

wereantibodies

subjected

againsttop94

Bioscience) coated Labtech chamber slides(anti-pIS2C antibody, 1:1000; further purified(Nalge Nunc International Inc.) or 60 mm

anti-pIS2 antibody (21) using the C-terminal

dishes (Asahi Techno Glass) in growthpart of the pKrich peptide (24), i.e.,

medium (20% FBS, and 0.2 μM ascorbic acid

NH2-VKKKKNKPIIFVC-COOH),(Wako Pure Chemical Industries Ltd.), inembryonic

Dulbecco’s

mediumDevelopmental Studies Hybridoma Bank(DMEM)). To induce muscle differentiation,(DSHB), University of Iowa), or desmin

the medium was switched to differentiation(1:1000; Sigma). An affinity-purified rabbit

medium (5% horse serum (Invitrogen), andanti-p94:ex15–16– antibody was generated

0.2 μM ascorbic acid in DMEM) for further

using

culture. All media were supplemented with

SVDRPVPRPGHT-C, corresponding to the

100 U/ml penicillin, 0.1 mg/ml streptomycin,

sequence encoded by transcript of exons 14

and 2 mM L-glutamine (Invitrogen).

and 17 of the mouse p94 gene. Subsequently,

RT-PCR analysis - Total RNA was preparedmembranesfromperoxidase-conjugated secondary antibodies

modifiedculturedmuscleEagle’s

cells

from

the

MHC

the

(cloneF1.652,KLH-conjugatedwere

peptideincubated(1:10;

15-week-old C57BL6 mice, using TRISOL

visualization of reacted bands using a POD(Invitrogen) according to the manufacturer’s

immunostaining kit (Wako Pure Chemical

instructions.

Industries Ltd).

strandcDNAwasInc.),

withquadriceps femoris and the soleus muscles ofFirst

Nichirei

1:50;

followedbysynthesized from 5 μg of total RNA using a

Immunofluorescent staining - Cultured cells

First Strand Synthesis kit (GE Healthcare

and muscle tissues were fixed and then

Bio-Sciences Corp.). Primers used to detect

stained as previously described (23). For

p94, splicing variants of p94, connectin, andmuscle tissues, longitudinal cryostat sections

μ-calpain are shown in Table I. The cycle(7 μm) of EDL muscles from six-month-old

number for PCR was 25 for all samples.

C57BL/6 mice were made. Antibodies used inWestern blot analysis - Cells were harvested

immunofluorescencein the presence of the homogenizing buffer

affinity-purified goat anti-pIS2C antibody(20 mM Tris/Cl (pH 8.0), 0.1 mM EDTA (pH(1:300),8.0), 1 mM DTT, 28 μM E64, 20 μg/ml(1:1000; Sigma), affinity-purified chicken

3

mouse

studiesanti-s-α-actinin

wereantibody

anti-connectin N2A antibody (1:500; (25)),

vivo. Thus, only s-α-actinin was further

affinity-purified rabbit anti-connectin M8M9

analyzed as to its interaction with p94.

antibody (1:300; (26)), rabbit anti-p94 NS

cDNA

antibody (1:300; (24)), and affinity-purified

mammalian cells - The cDNA corresponding

rabbit anti-pΔex15/16 antibody (1:300; thisto full-length human p94, mouse p94 variants

study). Alexa-488- or Alexa-555-conjugatedlacking exon 6 and/or exons 15 and 16

secondary antibodies were used (Molecular(p94ΔexX), and their protease-inactive forms,

Probes Inc., Eugene, OR). Nuclei were

p94:C129S (p94CS) and p94ΔexX:CS, were

stained(4,cloned into pcDNA3.1/N-FLAG vector (28).

6-diamidino-2-phenylindole dihydrochloride)

The NS domain of human p94 was cloned(Molecularwereinto pEGFP-C1 (Clontech). The cDNA for

analyzed on a laser scanning confocal

human s-α-actinin 2 (nt: 174–2921 of

microscopic system (LSM 510; Carl Zeiss

NM_001103, aa: 1–894) with an N-terminal

Inc.), which employed a Zeiss Axiovert

Myc-tag

inverted microscope with a Plan-Apochromat(Invitrogen).

63× (NA 1.4) lens. Images were recorded and

Immunoprecipitation - Expression vectors forprocessed with LSM510 imaging software.

Myc-tagged

Yeast two-hybrid screening - The cDNA

Flag-tagged human p94 or GFP-taggedsequence corresponding to the NS domain ofhuman p94 NS were transfected into COS7

human p94 (nt: 307–531 of NM_000070, aa:

cells. Cells were harvested 72 hr after

1–75) was cloned into pGBKT7 (Clontech).

electroporation and then homogenized in lysis

The bait plasmid and a human skeletal muscle

buffer (0.5% digitonin, 50 mM CsCl, 10 mM

cDNA library (HL4010AB, Clontech) were

Triethanolamine, 1 mM EDTA, 2 mM

cotransformed into Saccharomyces cerevisiae

phenylmethylsulfonyl fluoride, 10 μg/ml

AH109, according to the manufacturer’s

aprotinin,

instructions. About 9 × 10 transformants

centrifuged at 20,000 × g for 15 min, and thewere screened on selection medium plates,

supernatants preincubated with protein G

SD-LWHA, at 30°C, yielding 20 grownsepharose (GE Healthcare Bio-Sciences) tocolonies. Prey plasmids were isolated fromabsorb nonspecific binding proteins. After

these

with

TOTO3
Probes).

or

DAPI

Specimens

6colonies andsequencing.

Three

was

for

clonedhuman

pHexpression

7.8).

into

the

pcDNA3.1

s-α-actinin

Cell

in

and

lysates

were

to

DNA

centrifugation,

plasmids

wereincubated with anti-Myc antibody (9E10;

subjected
prey

constructs

supernatants

wereconfirmed for their reproducibility, two of

DSHB),ANTI-FLAG

M2

which encoded s-α-actinin (corresponding to(Sigma),or

antibody

aa 441–568 in ACTN2), and the other one

Clontech). After incubation with protein G

encoded von Willebrand factor (vWF). As

sepharose for 60 min, which was omittedvWF is an extracellular protein secreted bywhen ANTI-FLAG affinity gel was used,

endothelial cells (27), we did not further

immunocomplexes

analyze if vWF indeed interacts with p94 incentrifugation. The resin was washed with an4anti-GFP

wereaffinitycollected

gel(JL-6,

byexcess amount of lysis buffer followed byexamined (Fig. 1 A). Differentiation ofelution of immunoprecipitated proteins with

cultured cells was confirmed by decreased

SDS sample buffer.

expression of embryonic myosin heavy chain

Transfection into primary skeletal muscle(MHC) compared with the expression ofcells - Transfection was performed using

desmin (Fig. 1 B), and by the formation ofTransIT-LT1 transfection reagent (Mirus Inc.).

sarcomere

On the indicated day, cells were fixed andproliferating presumptive myoblasts (PM),analyzed by immunofluorescent staining.

i.e., cells before the onset of differentiation,

Quantitative

different

p94 and its splicing variants were hardly

sarcomeric localization was performed by thedetected (Fig. 1, C–F, PM). Predominating

followingamong the tested p94 splice isoforms,

comparison
formula:accumulation(%)

ofM-line

=

the

(N2A)

numberstructures(see

below).

In

transcripts for full-length p94 (Fig. 1 A, FL)ofsarcomeres which presented accumulation of

becameFlag-tagged(N2A

induction of muscle differentiation, and was

region)/total number of counted sarcomeres.

observed throughout muscle maturation (Fig.

Sarcomere length was measured as the

1, C–F, arrows). Splice isoforms lacking exon

distance between the centers of adjacent

6 (Fig. 1A, Δex6) were recognized from day 1

M-lines, which is positive for connectin

to 13 (Fig. 1D, open arrowhead). Splice

M8M9, using Zeiss LSM imaging software.

isoforms lacking exons 15 and 16 (Fig. 1 A,

Calculated ratios were resorted according toΔex15/16) were also observed from day 1 tosarcomerecells

13, but the amount relative to the transcripts

expressing p94WT, p94CS, p94Δex6:WT, andwith exons 15 and 16 (Fig. 1 A, ex15/16+)

p94Δex6:CS, 108, 107, 47, and 86 sarcomeres,was very low (Fig. 1 E, open arrowhead). The

respectively, were counted.

expression of variant transcripts without

p94

in

lengths.

M-linesIn

cultureddetectable

immediately

after

exons 6, 15, and 16 (Fig. 1 A, Δex6/15/16)
RESULTSwas slightly detected at day 3 (Fig. 1 F). All
tested adult skeletal muscles only expressed a

Transcription

of

the

p94

gene

duringfull-length

p94,

i.e.,

no

exon-skipping

myofibrillogenesis

variants were detectable (Fig. 1, C–F, QC andExpression of p94/calpain 3 and its splicing

Sol). In contrast, transcripts of the μ-calpain

variants during myofibril development was

large

analyzed

corresponding

expression in all samples (Fig. 1 G),

transcripts in primary mouse skeletal muscle

consistent with its proposed nature as acell culture. As previous studies have revealed

housekeeping gene (30).

bydetectingsubunitgene

showedconstantthat mouse embryonic muscles express p94
variants without exons 6, 15, and/or 16 (29),

Changes in the protein amounts of p94

the relative abundance of transcripts resulting

isoforms during myofibrillogenesis

fromNext, the expression of protein products

thoseexon-skipping

eventswas5corresponding to the p94 transcripts detectedstudies indicate that p94Δex isoforms areabove was examined. No 94 kDa full-length

expressed in immature myotubes, whereas

p94 protein was detected in cells cultured

full-length

under proliferating conditions (Fig. 1 H, PM).

throughout muscle differentiation.

Immediately

after

the

induction

p94

constantly

accumulatedofdifferentiation, full-length p94 was detected

Constant expression of the two p94 binding(Fig. 1 H, arrow), and the amounts of protein

sites

were

differentiation

notdownregulated

duringinconnectin/titinduringmusclemyofibrillogenesis. In addition, the amounts

p94 binds to connectin/titin at I82–I83 in theofmigrating

N2A region and at is7 in the M-line (Fig. 2 A)

bands—possible proteolytic fragments of(31,32). Thus, expression of these regions

p94—decreased,suggestingstabilization

p94

proteininthefasterthat

thewas also investigated in cultured skeletal

facilitatedasmuscle cells. N2A connectin was expressed at

myofibrillogenesis proceeds (Fig. 1 H, closed

all stages examined (Fig. 2 B). The 559 bp

arrowheads). In adult skeletal muscles, thetranscripts corresponding to the M-line region

predominating full-length p94 is coexpressedwith is7 (is7+) were also constantly expressedwith lower amounts of shorter isoforms or(Fig. 2 C). The 256 bp bands encoding

proteolytic fragments (Fig. 1 H, QC).

is7-lacking molecules and (is7–) were faintly

Based on molecular size, the 89 kDa banddetected in cultured cells. Consistent with

(Fig. 1 H, open arrowhead) could be variants

previous reports on muscle fiber typelacking either exon 6, 15 and/or 6, ordependent expression of is7 (12); both is7+

proteolytic fragments of full-length p94.

and is7– variants were expressed at similar

Therefore,levels in the quadriceps femoris, which

of

an

is

anti-pΔex15/16

antibody

specific for p94 isoforms lacking exons 15

predominantly

consistsof

fast

twitch+and 16 (Δex15/16) were raised by using a

myofibers, whereas is7 predominated in the

peptide spanning the exons 14 to 17 junction

soleus, composed of both fast and slowof the mouse p94 as an antigen (Fig. 1, A andmyofibers (Fig. 2 C). These results indicatedI). The antibody detected the 89 kDa bands

that the two p94 binding sites in connectin are(Fig. 1 I), and its protein amount graduallyalmost constantly provided during muscle

decreased during myofibril maturation.

differentiation.

contrasttotheresultsofIn

RT-PCR,

anti-pΔex15/16

antibody

recognizedtheCytosolic but not sarcomeric localization of

89kDa

in

suggestingthat

p94 during early myofibrillogenesis

band

PM,

p94Δex15/16 protein might not quickly be

To clarify when p94 is incorporated into

degraded and accumulate in PM cells evenspecific

though the corresponding mRNA expression

localization of p94 in cultured myotubes at

was extremely low (Fig. 1 C, E, and F).

different stages were analyzed. At day 3,

Together, our transcriptional and westerns-α-actinin was observed as punctuated6

positions

withinmyofibrils,

pattern, i.e. the I–Z–I bodies, in emerging

and M8M9, at their proper locations—Z,

growth tips where de novo synthesized

N2A, and M, respectively. p94 was still

myofibrillar proteins are assembled into

detected in the cytosol as granular structures,

sarcomere structures (14,33) (Fig. 3 A,

as seen at day 3, in more than 80% ofbrackets), and Z-band striations were seen in

myotubes, and, infrequently, the localization

the shaft of the same myotubes (Fig. 3 A,ofarrows). Conversely, using an anti-pIS2C

localization of connectin M8M9.

antibody, p94 was detected as small granules

At day 10, embryonic MHC was hardly

in the cytosol of whole myotubes without

detected in cultured cells by western blot

colocalization with s-α-actinin (Fig. 3, B, E,

analysis (Fig. 1 B), indicating that the cells

and H).

had become almost fully mature myotubes. In

The relationship between p94 and connectinthese mature myotubes, in addition to the

localization

staining

cytosolic distribution, two main types of p94

myotubes using two different anti-connectin

positive structures were observed. One wasantibodies. The anti-N2A antibody (antigen

M-line striations coinciding with M8M9

map in Fig. 2 A) detected grainy signals in thesignals (Fig. 3, P–R, arrowheads and arrows).

growth tips, and striated patterns in the shaft

The other was in the I-bands, possibly in theof some myotubes (Fig. 3 D, bracket andN2A regions (Fig. 3, M–O, brackets andarrows). An anti-M-line connectin (M8M9,

arrows). Intensity in the M-lines was stronger

antigen map in Fig.2A) antibody detected

than that in the I-band staining. Localization

striated signals in the shaft of myotubes, butof p94 in Z was not significant (Fig. 3, J–L).

none in the growth tips (Fig. 3 G, arrows andThese results indicate that p94 is incorporated

brackets). No colocalization of p94 with these

into myofibrils after the sarcomere structures

two regions of connectin was observed (Fig. 3,

are fully mature, through association withF and I). At this stage, p94 signals were also

above mentioned regions of connectin.

was

exploredbyp94

precisely

coincided

with

the

detected in myonuclei (Fig. 3, B and H). In
summary, p94 was not localized in theDistinct localization of p94 isoforms in

Z-bands including the I–Z–I bodies, in thesarcomeres

N2A regions, or in the M-lines at this stage,

As shown above, expression of p94Δex15/16

while s-α-actinin and connectin were already

was restricted in developing myotubes (seeorganized in striated patterns.

Fig. 1, E, F, and I). Thus, the localization of
p94Δex15/16 was examined in cultured

p94 is targeted to the N2A regions and the

myotubes at day 6 (Fig. 4, A–C). The

M-linesanti-pΔex15/16 antibody captured fuzzy thick

in

the

later

stagesofmyofibrillogenesis

striations, which almost overlapped with the

At day 6 of culture, more than 90% of

s-α-actinin signals at Z (Fig. 4, E and F,

myotubes showed typical myofibrillar protein

brackets), or doublets around Z (Fig. 4, H andlocalization, with s-α-actinin, connectin N2A,

I, arrowheads). These data indicate that7

p94Δex15/16 is probably localized to N2A or

stained with the anti-pNS antibody (Fig. 5 H,

somewhere between Z and N2A. In addition,

closed arrowheads and asterisk). In both cases,

p94Δex15/16 was found in M (Fig. 4 E, open

signal intensity was less in M (Fig. 5, E and H,

arrowheads). Contrary to the anti-pIS2C

open arrowheads). In summary, p94 isantibody results, the anti-pΔex15/16 antibody

localized in Z, N2A and M in both maturing

stained N2A/Z more strongly than M. These

muscle cells and skeletal muscle tissues. It is

results suggest that in developing myotubes,

noteworthy, however, that antibodies raised

not only the expression but also theagainst distinct regions of p94 showed

localization of p94 isoforms are regulated in adifferent propensity for staining in those three

way distinct from that for the full-length p94.

regions, suggesting that some proteolytic
fragments of p94 are associated with thesarcomere structure.

The p94 NS domain is localized in the
Z-bands
As shown previously, p94 was detected in Z,

s-α-actinin binds to p94 through the NS

especially by antibodies against the p94

regionN-terminusZ

To identify p94 binding molecules, yeast

localization of p94 was not detected in

two-hybrid (YTH) screening was carried out.

cultured skeletal muscle cells with the

A middle region of s-α-actinin (correspondinganti-pIS2C antibody. When the cells at day 6

to amino acids (aa) 441–568 in ACTN2) wasof culture were stained using the anti-pNS

identified from the human skeletal muscle

antibody, which was elicited against thecDNA library as a binding partner for the p94

N-terminus of p94 (NS region), the Z-band

NS region. Using a series of truncation

structure was detected in the shaft ofmutants of s-α-actinin, it was shown that therelatively mature myotubes (Fig. 5, A–C,

original clones identified by the screening

arrows and arrowheads), indicating that a p94

encoded the minimal binding site for the p94

fragment containing at least the NS region

NS region, which corresponds to the region

exists in Z. In contrast, much less, if any,

spanning the C- and N-terminal halves of thestaining at N2A and M was obtained with thissecondantibody. The anti-pNS antibody also detected

respectively (Fig. 5 J).

small granular structures in the cytosol ofAsmyotubes (Fig. 5 B), which were notmolecules of p94 and s-α-actinin was not

colocalized with s-α-actinin.

detected in YTH, coimmunoprecipitation

The localization of p94 in adult skeletal

analysismuscles was also examined with anti-pIS2C

s-α-actinin

and anti-pNS antibodies. Anti-pIS2C antibody

proteolytically inactive missense (C129S)

labeled preferentially N2A (Fig. 5 E, closed

mutant, were coexpressed in COS7 cells, andarrowheads), leaving Z less intensely stained

subjected to immunoprecipitation with either(asterisk), whereas both Z and N2A wereanti-Myc or anti-Flag antibody. Interaction(19,22).

However,

clear

8

and

third

interaction

was
and

spectrin-likebetweencarriedtheout.

Flag-tagged

repeats,

full-length

Myc-tagged
p94CS,awas

indicate that exogenously expressed p94 anddemonstrated in both immunoprecipitations

its variants are targeted to the same specific(Fig. 6, A and B). The p94 NS region fused toposition in the myofibrils as endogenous p94

GFP was also shown to interact withis, and that proteolytic activity was not

s-α-actinin (Fig. 6, C and D). These results

required for the process.

betweenp94

ands-α-actinin

indicate that p94 directly interacts with
s-α-actinin

via

the

NS

region,Stretch- and protease-activity-dependence ofwhichcomprises at least a part of the mechanism for

myofibrillar targeting of p94

its Z localization.

To compare the behavior of p94WT and
p94CS in a more dynamic aspect, theProtease-activity-independent

targeting

localization of exogenously expressed p94

ofp94 to specific positions in myofibrils

was evaluated in relation to sarcomere lengths,

It was of interest whether the proteolytici.e., the extent of contraction/extension.

activity and/or the molecular structure of p94

p94WT andaffects its targeting to the specific positions intargeted to M in short, contracted sarcomeres

the sarcomeres. Therefore, Flag-tagged p94(Fig. 7, B and H). In stretched sarcomeres,and its splicing variants with and without

they were more localized to N2A (Fig. 7 E).

proteolytic activity were expressed in cultured

p94

skeletal muscle cells to examine theirp94Δex6:CS (Fig. 7, K and N) showed the

localization. Six days after transfection, cells

same trends. Plotting the frequency of

expressing

detecting p94 in N2A or M against sarcomere

Flag-tagged

wild-type

p94

variants

p94CS

such

as

p94Δex6

andlengths

less than those observed for cells expressing

assessment; more than 50% of Flag-p94WT

protease-inactive Flag-p94:C129S (p94CS).

was detected in M when sarcomeres wereThis is possibly due to p94WT disappearing

shorter than 2.6 μm, while in the longer

by autolysis more rapidly than p94CS, and/or

sarcomeres,that the excess amount of expressed p94WT

predominant (Fig. 8 A). Interestingly, we

is cytotoxic for myotubes. Both p94WT andfound subtle but significant differences when

p94CS were mainly incorporated into thecomparing the stretch-dependent targeting of

M-lines (Fig. 7, B, E, and H, closed

p94CS with WT. For p94 CS, the thresholdarrowheads). Other p94 variants, such aslength was elongated to 2.8 μm (Fig. 8 B).

p94Δex6, p94Δex15/16, p94Δex6/15/16, with

Similarly, for p94Δex6, higher stretch levels

and without the CS mutation, showed trends

were required for redistribution from M toessentially identical to those of p94WT and

N2A for the CS mutant (2.2 vs 2.6 μm; Fig. 8,

p94CS

Myotubes

C and D). In summary, our results indicate

expressing excess amount of p94CS and otherthat p94 targets to M in relaxed and/or

inactive variants did not show any defect incontracted myofibrils, and is redistributed

assembling striated myofibrils. These results

into N2A by a stretch-dependent mechanism.

data).

9

N2A

more

preferentially(p94WT) gave signals one-third to one-tenth(unpublishedallowedwerequantitative

localizationwasTargeting into N2A requires higher amounts

found in N2A, but no p94 isoform wasof stretch for the proteolytically inactive p94

detected in the sarcomeres (IV). Previously,

mutants.

the IS2 epitope was detected at both Z and
N2A in adult myofibrils (19), which isdifferent from the results shown here, that is,

DISCUSSION

localization mainly at N2A and faintly at M.
In this study, the distinctive features of theThis is probably because a more specific,

expression and localization of p94 and its

affinity-purified anti-pIS2C antibody was

splicing variants during myofibrillogenesis in

used in this study.

the primary culture of skeletal muscle cells,
and a novel interaction between the p94 NS

Regulation of p94 in the cytosol on its

regionautolysis and targeting to Z/N2A/M

ands-α-actinin

areshown.

Furthermore, we have shown that thep94 targets to Z/N2A/M after the assembly of

localization of exogenously expressed p94

basic sarcomere components. By day 3 ofchanged in correlation with sarcomere length,

differentiation of cultured myotubes, clear

with p94 proteolytic activity required for thisstriation of Z, N2A, and M, containing

response. This potentially indicates thats-α-actinin and connectin, is detected, but p94

cellular localization of p94 and possibly of its

is predominantly distributed in the cytosol asproteolytic

small granules. This observation raises atactivityisregulatedinastretch-dependent manner.

least

Fig. 9 A summarizes p94 localization

investigation. One is what molecules otheraccordingofthan unproteolyzed p94 are contained in thesemyofibrillogenesis. I: in the early stage, I–Z–I

granules, as the signals—detected by bothandanti-pNS

thick

to

developmental

filaments

arestagesindependently

twoquestionsandworthy-pIS2Coffurther

antibodies—wereday 6, fundamental

distinct from the diffuse staining of soluble

myofibrillar components are incorporated into

enzymes. Another is how targeting of p94 to

sarcomeres; III: in the late stage), myotubes

Z,

are fully mature; IV: adult skeletal muscle

developmental

comprises mature myofibers. Both p94 and its

molecules of p94 have already existed andisoforms (p94Δex) are distributed in the

been assembled into sarcomere structures.

cytosol with no myofibrillar localization (Fig.

One reason could be that there is transitional

9A, I). Then, some Z and M localization issteric hindrance masking binding sites whenobserved, although the majority of the protein

many different sarcomere components areremains cytosolic (II). Later, p94 isoforms are

dynamically incorporated into myofibrils.

mainly targeted to N2A, while IS2- andAfter day 6 in culture, it is notable that two of

NS-containing

respectively

the p94 regions, NS and IS2, showed distinct

accumulate in M/N2A and Z (III). In adultlocalizations; NS was almost exclusively in Z

tissues, both NS- and IS2-containing p94 iswhile IS2 was in N2A and/or M. One

assembled;

II: byfragments

10

N2A,

or

M

is

stage,

protected

at

even

binding

if

thisinterpretation of these results is that the N-

development, it is tempting to consider thatand C-terminal fragments were generated by

the interaction between p94 and p94Δex isproteolysis, presumably by autolysis, andone of the mechanisms that regulates p94, intargeted to Z and N2A/M, respectively. It isboth its protease activity and targeting. Later,

also possible that the affinity to Z or N2A/M

in mature muscle cells, where sarcomere

of NS or the respective remaining fragments

structures gain more integrity and complexity,

could be much stronger than that ofother mechanisms might allow rapid targetingfull-length p94. Data showing that NS bindsof p94 to myofibrils and/or suppress its

to s-α-actinin and that the region close to IS2

autolysis until being targeted to myofibrils.

interacts with N2A connectin (21) areconsistent with this idea.

Regulated expression of p94Δex during

In the same day 6 culture, exogenously

muscle development and its relevance

expressed Flag-p94WT and -p94CS showed

One of the p94 variants, p94Δex15/16, wasalmostinshown to be transiently expressed andN2A/M rather than in Z. This suggests that

predominantly incorporated into N2A only in

p94 proteolytic activity is not required for p94

immature myotubes, but not in adult skeletal

targeting, and that p94 molecules detected atmuscles.

N2A/M

IS2

p94Δex/p94 ratios at Z, N2A, M, and in theregions—these are undegraded p94 molecules.

cytosol are schematically shown in Fig. 9 B.

It may be possible that the affinity of NS to Z

Interestingly, we detected larger amounts ofis blocked by the extra Flag-tag sequence

p94Δex at N2A than at M, whereas full-length

allowing

nonproteolyzed

p94 is predominantly at M. This may indicate

Flag-p94 to be targeted to N2A/M. Still, therethat p94Δex have higher affinity to N2A thanshould be a mechanism in the cytosol so that

M at this stage. It is possible that theexogenously

disappearance of p94Δex from the N2A site

identical

localization—mainly

possesses

most

of

both

the

expressed

NS

p94

and

does

not

Developmental

changesofexhaustively autolyze.

enables p94 to gain access to it, which is

In more mature myofibrils—at day 10 inimportant for myofibrils to mature fully.

culture, and in adult skeletal muscle—NSIn

and/or IS2 epitopes become detectable in Z,

p94Δex6 or p94Δex15, but not full-length

N2A, and M. Thus, it is suggested that mature

p94WT,

muscle cells facilitate more efficient targeting

myofibers (34). These variants, which wereof nonproteolyzed p94 to those three sites inshown to be less active than p94, especiallythe sarcomeres.

with regard to autolytic activity (29), could

Altogether, these results suggest the existence

act detrimentally at various developmentalof unidentified molecules/mechanisms thatstages. Thus, these previous and our data are

regulate the activity of p94 in the cytosol. As

in support that both the transient expression

p94Δex proteins are expressed in amounts

and the disappearance of p94 isoforms at thesimilar to p94 at the early stages of myotube

early stages of myogenesis are important for11transgenic

mice,

overexpression

blocked/delayed

maturationofofmyotubes maturation, probably because thesesuch as α-actin (38), the N-terminal Z-repeat

isoforms respond differently to the cellularregion of connectin (15,16), myopalladin (39),mechanisms regulating p94. The differences

FATZ1/calsarcin-2/myozenin-1(40),

in the threshold sarcomere lengths for thecalsarcin-3/myozenin-3

andshift of localization from M to N2A—2.6 andcypher/ZASP/Oracle (42). Our finding of

2.2 μm for p94WT and p94Δex6 WT,

interaction between NS and s-α-actinin

respectively—could be one indication ofsuggests that p94 also contributes to thethese effects.

integrity of Z. Consistently, previous studies(41),

showed that p94 antisense oligonucleotide
Interaction between p94 and s-α-actinin

treatment disrupted Z-band structures andBy YTH screening, the middle region ofdiminished s-α-actinin detection (43). As the

s-α-actinin, which encompasses the junction

NS but not IS2 epitope was preferentiallyof two consecutive spectrin repeats and binds

localized in Z during myofibrillogenesis,to the NS region of p94, was identified as the

N-terminal proteolyzed fragment(s) of p94

long-sought binding partner for p94 in Z.

could be important for this aspect.

Direct interaction between s-α-actinin and NS,
as well as full-length p94, was detected in

p94 localization is affected by the sarcomere

COS7 cells. In yeast, however, the full-length

length and its proteolytic activity

s-α-actinin did not bind to p94, probably

The dynamic and distinct localization of

because full-length s-α-actinin tightly forms

exogenously

homodimers (35) and/or YTH sometimes

sarcomere length suggests that p94 senses

shows false negative results.

It should be

myofibrillar stretch—when sarcomeres arenoted that s-α-actinin showed a complicatedbecoming overextended, some portions offashion when binds to p94; i.e., s-α-actinin

p94 may change localization from M to N2A

fragments (aa401-894; aa430-894) did not(Fig. 9 C). In YTH, binding between p94 andbind to p94 NS region, even though theseconnectin is much stronger in N2A than in M

fragments contain the region able to bind p94(29), and the N2A p94 binding site is close to(aa441-568). Similar to our results, it was

extensible

reported that s-α-actinin showed intricate

connectin Thus, extension of connectin may

patterns when interacting with connectin (36).

unmask N2A epitopes important for p94

Therefore, although further study isrequired for determination of the precise
p94 binding site in s-α-actinin, minimum

binding, allowing p94 to be recruited from M

p94 binding region so far determined is theThis idea is supported by Trombitas’s study,

C-terminal half of R2 plus the N-terminal half

which showed that the change of sarcomereof R3 (Fig. 5K, red or pink region).

length up to ca. 2.5 μm is mainly by changes

S-α-actinin is one of the major components ofin IG structures, major motifs in N2A, andZ (37), and has multiple binding molecules,

further stretch is by extension of PEVK (44).

expressed

springp94

elements

relative(PEVK)

toof

to N2A and suggesting involvement of p94 insignal transduction pathways in myofibrils.

12Interestingly,

activeredistribution

explanation;ofmyofibrillogenesis

occursT-cap/telethonin, one of the connectin ligands,

normally, but with misaligned thick filamentsfrom M to Z has also been suggested as ainconsequence of its phosphorylation by theremodeling, which is induced by unloading

connectin kinase domain (ttk) (45). As

muscles, is impaired (47). Together with theconnectin

oppositelate incorporation of p94 into myofibrils,

half-sarcomeres overlap in M, both thethese data suggest that p94 participates in the

C-terminal is7 and ttk locate in the periphery

maintenance of sarcomere structures, whichof the M-line lattice (46). Thus, dissociation

undergo deformation while the muscle isof p94 from M potentially can be linked bycontinuously used.

stretch-activated phosphorylation mediated by

The most important question should be; whatttk.

“signalis p94 protease activity required for? Onethatpossibility is that it allows swift changes inconnectin-binding proteins in Z, N2A, or M

p94 structure by autolysis. Autolysis of p94

function as a complex at each position tohas been shown to proceed by “nicking” inmaintain cooperativity among the many

NS, IS1, and/or IS2 (21), with the reaction indifferent levels of muscle components, e.g.,

IS2 regulated by N2A. These proteolytic

between the sarcomere and the nucleus (32),modifications of p94 itself possibly result in awhere the extension/contraction states in N2A

more “movable” conformation for p94,

would be communicated to other levels by thealtering its affinity to connectin and otheraction of p94.

molecules.

Localization of p94CS and p94Δex:CS failed

exogenously expressed p94 and p94Δex6,

to reflect extension of sarcomere as promptly

which show different autolytic profiles (29),

as

This

were slightly different in their propensity toobservation is significant in that it relates, forchange localization between M to N2A. At

the first time, a defect in the proteolytic

present, one explanation for the sluggish but

activity of p94 to its behavior within the

not absent response of p94CS—devoid ofphysiologicalautolytic

Ourfilaments

previously

complex”

their

from

proposedhypothesizes

activecounterparts

event

do.

of

musclethesarcomeres(6),

Consistentactivity—to

and

with

sarcomere

thissarcomereidea,

lengthextension/contraction. Our results indicate

changes is that in our cultured myotubes,

that protease-defective p94 cannot initiate or

endogenoustransfer the cellular response required for

intramolecularly proteolyze p94CS. This issaving muscle from the enormous stresses

consistent with previous reports that p94 null

posed during cycles of extension/contraction,

mice showed more severe phenotypes thanwhich possibly leads to accumulated damagetransgenic mice overexpressing p94CS didof muscle, resulting in a muscular dystrophy(5,7). To provide more insight into thephenotype after some decades. Reported

physiological functions of p94, what property

phenotypes in skeletal muscles of p94 nullof p94 is involved in its response tomice

sarcomere conditions and how it is achieved

areconsistent

with

the

above

13

wild-type

p94

can

should be elucidated.
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FOOTNOTESWe are grateful to Tatsuya Maeda, Muriel Herasse, and Isabelle Richard for generous gifts of cDNA
constructs, and to Takuro Arimura, Akinori Kimura, and all of the Calpain PT members forexperimental support and valuable discussion. This work was supported in part by16

MEXT.KAKENHI 16026209, 17028055, and 18076007 (to HS), JSPS.KAKENHI 18700392 (toKO), 18770124 (to YO), and 18380085 (to HS), Research Grant (17A-10) for Nervous and Mental
Disorders from the Ministry of Health, Labor and Welfare (to HS), Sasagawa Scientific Research
Grant from The Japan Science Society (to KO) and the DFG (La668/7-2) (to SL).
The abbreviations used are: IS1 and IS2, specific insertion sequences 1 and 2 of p94; MHC, myosin
heavy chain; NS, N-terminal specific sequence of p94; p94CS, p94:C129S protease-inactive mutant;
PEVK, Pro, Glu, Val, and Lys-rich spring elements of connectin; PM, proliferating presumptive
myoblast; s-α-actinin, sarcomeric α-actinin (α-actinin 2); ttk, titin/connectin kinase domain; YTH,
yeast two-hybrid.
FIGURE LEGENDS
Figure 1. Analysis of the expression of p94 and its splicing variants during myogenesis. (A) A
schematic domain structure of p94, the antigens for the antibodies, and the exon structures amplified
by RT-PCR. NS, IS1, and IS2 are p94-specific insertion sequences. Domains IIa+IIb, III, and IV, arethe conserved calpain domains. Most of IS1 and IS2 are encoded by exon 6 and exons 15 and 16,
respectively. The expected sizes of the RT-PCR products are shown. (B) The expression ofembryonic MHC and desmin were analyzed by Western blotting. As skeletal muscle differentiation
proceeds, the expression of embryonic MHC relative to desmin decreases. (C–F) RT-PCR analysisof p94 transcripts using primer sets for amplification of exons 1 to 24 (C), 4 to 9 (D), 10 to 17 (E),and 4 to 17 (F). Arrows and open arrowheads correspond to the transcripts with no exons skipped
and those with exon skipping, respectively (C to F). Note that the skipping of exon 6 or exons 15
and 16 were not discernible from each other in transcripts covering exons 4 to 17 (F, upper open
arrowhead). (G) Stable expression of the μ-calpain large subunit during myogenesis. (H) Detectionof p94 protein in cell lysates using a p94-specific anti-pIS2C antibody. Full-length p94 and its
proteolytic fragments are indicated by the arrow and closed arrowheads, respectively. The open
arrowheads indicate p94 fragments, which could be p94Δex6, 15 and/or 16. (I) Expression of
p94Δex15/16 protein during differentiation detected with an affinity-purified anti-pΔex15/16
antibody. The open and closed arrowheads indicate p94Δex15/16 and its possible proteolytic
fragments, respectively. Lanes in panels (B)–(I): H2O, negative control; PM, proliferatingpresumptive myoblasts; Day 1, 3, 6, 10, and 13, cells differentiated for the indicated number ofdays; QC, Sol, and TA, quadriceps femoris, soleus, and tibialis anterior muscles, respectively from a
15-week-old C57BL6 mouse; p94:CS and p94:ex15–16–, proteins recombinantly expressed in COS7
cell.
Figure 2. Expression of transcripts corresponding to the p94 binding sites of connectin in the
N2A and M-line regions. (A) A single connectin molecule spans from the Z-band to the M-line.

17

The locations of the amplified regions in connectin by RT-PCR are schematically represented insarcomere structure (bars). The double-headed arrows correspond to the p94 binding sites. The
antigens for the anti-connectin N2A and M8M9 antibodies are indicated by brackets. I and M,
together with the numbers, indicate the immunoglobulin motifs in the I-band and the M-line,
respectively; PEVK, kinase, and is7 indicate the PEVK region, the kinase domain, and the insertion
sequences, respectively. Expression of transcripts corresponding to the p94 binding sites in the N2A
region (B) and the M-line (C) was analyzed by RT-PCR. The arrows and the open arrowhead
indicate products corresponding to the transcripts with and without the p94 binding region as shownin (A).
Figure 3. Colocalization of p94 with connectin at the N2A region or the M-line in the late but
not in the early stage of myofibrillogenesis. Cultured skeletal muscle cells at day 3 (A–I) and day
10 (J–R) were stained with the anti-pIS2C antibody (B, E, H, K, N, and Q) in combination with oneof the antibodies against s-α-actinin (A and J), connectin N2A (D and M), or connectin M8M9 (Gand P). Nuclei were stained with TOTO3 (blue; A-I). Merged images are shown at the right (C, F, I,
L, O, and R). Brackets indicate the areas of the growth tips. At day 3, s-α-actinin showedstress-fiber-like structures in a growth tip and the Z-band striation (arrows) in the shaft of myotubes(A and C). N2A (D and F), and M-line (G and I) striations were observed in the shaft of myotubes(arrows). p94 was detected in a granular structure in the cytosol and was not colocalized with
s-α-actinin, and connectin N2A and M-line regions (A–I). p94 signals were also detected inmyonuclei (B, C, H, and I). At day 10 myotubes, arrowheads and brackets indicate p94 localization,
whereas arrows indicate the localization of s-α-actinin (J and L), connectin N2A (M and O), andconnectin M8M9 (P and R). Insets show magnified images (J–R). Bars, 10 μm.
Figure 4. Localization of the p94 isoform p94Δex15/16 in the sarcomere.
In cultured myotubes at day 6, localization of p94Δex15/16 was examined using an anti-pΔex15/16
antibody (B, E, and H) in combination with an anti-s-α-actinin antibody (A, D, and, G). Merged
images are shown at the right (C, F, and I). D–I are these images at higher magnification.
Localization of p94Δex15/16 was observed as thick stripes at the Z-bands (E and F, brackets), as
dotted lines between the Z-bands (E and F, arrowheads), and doublets around the Z-bands (H and I,
closed arrowheads). p94Δex15/16 also accumulated as small particles in the cytosol of the shafts ofjuvenile myotubes (asterisk). Arrows indicate the Z-band localization of s-α-actinin. In the merged
image, nuclei (blue) were stained with DAPI (C). Bars, 10 μm.
Figure 5. Z-band localization of p94 and its interaction with s-α-actinin at the NS domain.
(A–C) Localization of p94 was examined in cultured myotubes at day 6 using an anti-pNS antibody.
p94 colocalized with s-α-actinin at Z (arrowhead, B and C). (D–I) In longitudinal cryostat sectionsof EDL muscles from six-month-old C57BL/6 mice, p94 was detected with anti-pIS2C (E) or

18

anti-pNS (H) antibodies, and Z was detected with an anti-s-α-actinin antibody (D and G, arrows). In
adult muscles, in contrast to day 6 myotubes, p94 was observed in both N2A and M (E, F, H, and I,
closed and open arrowheads, respectively) with both anti-pIS2C and anti-pNS antibodies. p94 wasalso detected in Z using an anti-NS antibody (H and I, *), but was less clear with anti-pIS2C (E, *).
Bars, 10 μm. (J) A schematic for the domain structure of s-α-actinin and its interaction with p94 NS
as detected by YTH assay. S-α-actinin consists of an N-terminal actin-binding domain, four
spectrin-like repeat domains, and tandem EF-hand motifs in the C-terminal region. The p94 NS
binding site in s-α-actinin was located between the centers of the second and third spectrin-like
repeat domains (aa: 441–568; double-headed arrow). (K) Ribbon presentation of the s-α-actinin
spectrin-repeat region. The two antiparallel subunits of the s-α-actinin spectrin-repeat region are
depicted in blue and green. Minimum p94 binding region (aa441-568) is shown in red or pink.
Ribbon diagrams were generated using MolFeat Ver.2.2 3D imaging software (FiatLux Inc., Tokyo,
Japan).
Figure 6. Interaction between p94 and s-α-actinin in mammalian culture cells. (A and B)
Myc-tagged s-α-actinin and Flag-tagged p94CS coexpressed in COS7 cells were examined for theirinteraction. Myc-tagged s-α-actinin was coimmunoprecipitated with Flag-tagged p94CS (A,arrowhead). Alternatively, Flag-tagged p94CS was detected in the sample immunoprecipitated withthe anti-Myc antibody (B, arrowhead). Coimmunoprecipitation of GFP-tagged p94 NS domain and
Myc-tagged s-α-actinin was also detected (C and D, arrowheads). Therefore, the interaction between
p94 and full-length s-α-actinin was shown to occur via the p94 NS region in mammalian cells. IP,
immunoprecipitation; WB, western blot.
Figure 7. Dynamic translocation of exogenously expressed p94 in relation to sarcomere
condition. At day 6 after transfection, myotubes expressing either Flag-tagged p94WT (A–C, G–I),
Flag-tagged p94CS (D–F), or Flag-p94Δex6:CS (J–O), were doubly immunostained with anti-Flag(B, E, H, K, and N) and anti-M8M9 antibodies (A, D, G, J, and M). Merged images are shown at theright (C, F, I, L and O), with nuclei stained with DAPI shown in blue. Transfected myotubes (G–O)
showed hyperextension (right side in G–I, and left side in J–O) and hypercontraction (left side in
G–I, and right side in J–L). (M–O) High magnification views of the border areas (J–L, rectangle) are
shown. Closed arrows indicate M. Closed and open arrowheads indicate signals for exogenously
expressed Flag-p94 at M and N2A, respectively. *, untransfected myotubes. Bars, 10 μm.
Figure 8. Quantitative analysis of the M-line and N2A localization of exogenously expressed
p94. At day 6 after transfection, the localization of Flag-p94WT, Flag-p94CS, Flag-p94Δex6:WTand Flag-p94Δex6:CS was evaluated (see Materials and methods). The sarcomere length between
the centers of connectin M8M9 positive signals corresponding to the M-lines was measured using
Zeiss LSM imaging software. Black and gray bars indicate the ratios of Flag-p94 localization in the19M-lines and the N2A regions, respectively.
Figure 9. Expression and localization of p94 in the context of myofibril development andsarcomere function. (A) Developmental changes in the expression and localization of p94 and its
isoforms are summarized. I) At the early stage of myofibrillogenesis, the N- and C-termini ofconnectin are associated with I-Z-I bodies and thick filaments, respectively. At this stage, p94 and its
isoforms are distributed in the cytosol (Cyt) as small punctuate structures. II) The sarcomere scaffold
is assembled, that is, interdigitating Z, N2A and M structures are formed. Most of the p94 remains in
the cytosol, with some populations of p94 and isoforms targeted to Z and/or N2A/M. III) Later, p94
is incorporated into Z, N2A, and M. p94 isoforms tend to be targeted to N2A rather than to M. IV)
In adult skeletal muscles, p94 isoforms were absent, and p94 localizes in Z/N2A/M. The structuresof p94 and its isoforms are schematically shown; the gray domain might be proteolytically removed.
Localization and relative ratios of p94 and its isoforms at specific regions, and the signals with eachantibody are qualitatively summarized. (B) Qualitative profiles of p94Δex/p94 ratios in the four
positions during myofibrillogenesis as discussed in (A) is shown. (C) A proposed model of p94
translocation in response to over and under stretching of muscle. In the range of normal
contraction–stretching conditions, exogenously expressed p94 is located in the M-lines. Whensarcomeres are over stretched, p94 accumulates in the N2A regions, being released from the M-lines
and/or recruited from cytosol.

20

Table I. Primers for RT-PCR detection of p94, splicing variants of p94, connectin/titin, andμ-calpain

Primersense (5′–3′)

antisense (5′–3′)

p94 Exon 1 atcgagctcggatcc-ATGCCAACTGTTATT–Exon 24
p94 Exon 4–Exon 9p94 Exon 4–Exon 17
p94 Exon 10–Exon 17AGT*
AACCACCGCAATGAGTTCTGG
AACCACCGCAATGAGTTCTGG
ATAAGCTTCAGACCTGGACG

GGTTCAGGCATACATGGTTGTCCACAAAGCTCCAGTCC
tcccccgggtca-TTGTTGCTGTTCCTCA
CTTTCC*tcccccgggtca-TTGTTGCTGTTCCTCA
CTTTCC*

connectin/

AAGGTACCAACCACTGCCACGTTT

AAAGGATCTTTTTTCACACCCTTT

titin N2A

ATTGCAATTGGTCACGTGTACACTCTTGAAATCC

CTTCCTTGTTGTTCTTGGTTTTGG

GAATTGGAATACCACATTTTACGATCAAAGGTCACAACACCATCCAG

GG

Gconnectin/
titin M9M10
μ-calpain*These primers are as same as used in (48). The additional unrelated sequences were for cloning
purposes.

21

Figure 1 (Ojima, K. et al.)

B
eMHC

pNS

p94 NS
exon

IIa

pIS2C
IS1 IIb

III

6

IS2
15,16

C
2,480 (FL)exon 1 - 24

exon 4 - 9

Desmin

IV

(bp)
4,361
2,322
2,027

M
ar
H ke
2O r
PM
D
a
D y1
a
D y3
a
D y6
a
D y1
a 0
Q y1
C 3
So
l

Antigens ofantibodies

PM
D
ay
D 1
a
D y3
a
D y6
a
D y 10
ay
Q 13
C

A

D

556 (ex6+)

600
517
400

exon 10 - 17688 (ex15/16+)

exon 4 - 17

1,376 (FL)

E
700
600
517

F
1,517
1,200
1,000

G
1,200
1,000

97

66

66

53
45

40
30
anti-pIS2C

4C
TA S
So
l

p9

ay
D 1
ay
3
D
ay
D 6
ay
D 10
ay
13

(kDa)

97

D

(kDa)

PM

p9
4C

S

I
PM
D
ay
D 1
ay
D 3
ay
D 6
ay
D 10
ay
Q 13
C

H

Figure 2 (Ojima, K. et al.)

A

Z

M

Z
Thin filament
Thick filament

N2A

Connectin/titin

p94 binding site

p94 binding site
I80

I81 I82 I83

Antigens ofantibodies

kinase

PEVK

M1

M2 M3

M4

M5

pN2A

M6

M8 M9 is7 M10

M7

pM8M9
559 bp (is7+)
256 bp (is7-)

1,453 bp (N2A)

1,500
1,400
1,300

(bp)

N2A

600
517
400
300
200

So

l

13
C
Q

10

ay
D

6
D

ay

3

ay
D

1

ay
D

ay
D

PM

k
H er
2O

M

So

l

ar

13
C
Q

10

ay

D

D

6
ay

ay

3
ay

D

1
ay

D

D

ar
M
(bp)

PM

C
k
H er
2O

B

is7+

is7-

Figure 4 (Ojima, K. et al.)s-α-actininAp94∆ex15/16

B

merge

C

*
D

E

F

G

H

I

Figure 5 (Ojima, K. et al.)s-α-actinin
A
B

merge

p94

C

pNS
D

E

F

*

*

pIS2C
G

H

I
*

*

pNS
J

1

200
CH
DomainCH
DomainActin Binding Domain

400
R1

600
R2

R3

740 800R4

894 (aar)Binding to
p94 NSE E
F FSpectrin-like Repeat Domains-α-actinin (1-894)
s-α-actinin (303-894)
s-α-actinin (344-894)
s-α-actinin (356-894)
s-α-actinin (401-894)
s-α-actinin (430-894)
s-α-actinin (517-894)
s-α-actinin (614-894)
s-α-actinin (690-894)
s-α-actinin (441-568)s-α-actinin (405-505)s-α-actinin (520-627)

K

R4

R3

R2

+
+
+
+
-

R1
C
N

C
N

R1

R2

R3

R4

Figure 6 (Ojima, K. et al.)A

B
LysateMyc-s-α-actinin
Flag-p94CS

IP: anti-Myc

IP: anti-Flag- - + + - - + + - - + +- + - + - + - + - + - +LysateMyc-s-α-actinin
Flag-p94CS(kDa) 97
67(kDa) 9745

45

IP: anti-Flag

IP: anti-Myc- - + + - - + + - - + +- + - + - + - + - + - +67

WB: anti-Myc

C

WB: anti-Flag

D
LysateMyc-s-α-actinin
GFP-NS
GFP(kDa)

IP: anti-Myc

IP: anti-GFP- - + + - - + + - - + +- + - + - + - + - + - ++ - + - + - + - + - + -LysateMyc-s-α-actinin
GFP-NSGFP

IP: anti-GFP

IP: anti-Myc- - + + - - + + - - + +- + - + - + - + - + - ++ - + - + - + - + - + -(kDa) 67

97
67

45

45
30
WB: anti-Myc

WB: anti-GFP

Figure 7 (Ojima, K. et al.)

M8M9

A

Flag-p94

merge

B

C
WT

D

E

CS F

G

H

I

*

*

J

K

*

*

WT

L

*

*

∆ex6:CS

M

NO

Figure 8 (Ojima, K. et al.)

A (%)

C (%)

100
80

100

WT

80

60

60

40

40

20

20

0
100
B

0
100
D

CS

80

80

60

60

40

40

20
0 .6 .8 .0 .2 .4 .6 .8 .0 .2 .4 .6 .8-1 -1 -2 -2 -2 -2 -2 -3 -3 -3 -3 -3
1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
Sarcomere length (µm)

M-lineN2A∆ex6:WT∆ex6:CS20
0 .6 .8 .0 .2 .4 .6 .8 .0 .2 .4 .6 .8-1 -1 -2 -2 -2 -2 -2 -3 -3 -3 -3 -3
1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
Sarcomere length (µm)

Figure 9 (Ojima, K. et al.)A

~ Day 3~6

~ 10

Immature

Sarcomere
localization

I-Z-I
body

Cyt

Z N2A M

Cyt

Z

pNS

-

++

++

-

-

++

++

pIS2C

-

++

-

+-

+

++

-

++

-

++

+-

++

NS NS

NS

NS

Antibody

Sarcomere
integrity

NS

NS

adult
tissueMature

NS

NS

N2A M

Cyt

Z N2A M

Cyt

+-

-

+

++ ++

+-

+-

-

+

++

+

+-

++

+

+-

+-

+

+-

+-

-

-

-

-

NS NS

NS

NS

NS

NS

NS

IS1

IS1

IS1

IS1

IS1

IS1

IS1

IS2

IS2

IS2

IS2

IS2

IS2

IS2

Z

Z

M
N2A

I

II

III

B

IV

Thick filament

Immature I-Z-I bodyThin filament

Z-band

IS2

NS

1

IS1

Connectin/Titin

N2A

,

p94

NS

Developmental
stage

,

Cyt

M
Z
0

I

II

III

IV

Developmental stageC

Normal contraction/stretch

Over-stretched?
?

?