Vol.

Ta JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY Copyright © 1975 by The Histochemical Society, Inc.

STRUCTURE

OF

MUSCLE

FILAMENTS ULTRASTRUCTURAL

AND

FRANK Department

In

of Anatomy,

this

presentation

muscle

filaments

traction,

that

ments.

the

are thin

thick

I will

School,

I will

which is, the

and

ments

Medical

deal

deal

antibody-labeling

with

the

with

methods

in

the

of these

filaments.

the is actin

structural and of

protein the thick

of the filament

myosin,

other

proteins

are

associated

tropomyosin

and

are

The

proteins

associated

and

they

tween

actin

present filaments. in the

with

actin

in the

to

regulate

the

serve and

myosin

along The thick

(81).

thin

the entire length proteins associated

filament

are

actin is

troponin

an

how

are

can

review)

propriate

briefly

along

the

filament.

The

teins is presently Antibody-labeling

the

ence

have

microscopy

to

specific

function

object studies

of these

pro-

primarily

involved

has

popular

strands

tered

37).

antibodies

tagged

In electron (46-48,

antibodies

fully

used

cle.

The

for use

(44,

microscopy,

55, 57, 66)

58)

have

and been

antibody-labeling

studies

of

antibodies

ferritin-tagged

sents a difficulty in the study of striated in that the tagged antibody is probably to penetrate ments

(50).

peroxidase-tagged yet

been

used

the actin and

between To

my

knowledge

antibodies in studies

that of

can anti-

been

on

verte-

in detail,

I will

features

filaments

of’ the

in vertebrate

and

cells. The similar

studied.

The

helically

wound monomers.

cross

studies

structure

of

in all

cells

filament

is made

strands

of

The

over

about

and

there

the actin in which it up

helically

wound

A

360-370

every

of

essentially along

ferritin-

the

success-

monomer

units

pomyosin long and

is a rod-shaped molecule about 400 A lies in the grooves between the two

of muspremuscle too large

at

The

troponin,

can

brate

be

of the

Institute. 2 Presented at the Symposium on Histochemical Approaches to Microfilaments at the 26th Annual Meeting of The Histochemical Society, April 13, 1975, Atlantic City, N. J.

similar

muscle striated

and

:380

14 Tro-

molecule,

A

(6).

vertebrate

striated

are

(18,

have

is a smooth

filament

and

been

The

Downloaded from jhc.sagepub.com by guest on March 13, 2015

esti-

in vertefilaments

in

are structurally 1.5 z in length

portion

a rough

543

No

51). in

reported.

myosin

muscles

all about

There

51).

of act in filaments

filaments:

vertebrate

(14).

globular

about

in

13 and

repeat

considerable

length

smooth

Myosin

between

little variation in length (45, striated muscles the variation

In invertebrate

Health Muscle

a of

filaments

show

length

are

cross-over

intervals

actin

muscles

not

per

with

present

fila-

have

filament

strands

of muscle.

was supported by U.S. Public HL15835 to the Pennsylvania

problems

structural

muscle

filaments: is very

mates 1 This work Service Grant

cases,

the molecWhere ap-

interpretation

filaments

the

myosin

spherical

unal-

myosin 39)

and

two

horseradish (38,

compare

Actin filaments

use of fluorescein as the fluorescent tag. Although other fluorescent tags have been used in antibody-labeling studies, fluorescein is most (10,

muscle

invertebrate

the

in specific

the

not

(see reference 50 attempt to show

antibody

discussing

striated

actin

of speculation. of muscle in fluores-

have

results.

Before

regions

out the

considI will

about filaments.

of the in

dis-

to the to the

since

filament.

studies,

encountered

brate

techniques and then

review but will

point

antibodyI will

studies

information

I will

filament

limited

from

detail

myosin

earlier

and

filaments

studies.

antibody-labeling

be

myosin

greater

the

exhaustive

body-labeling

are

on

yield detailed ular organization

(5, 34,

and muscle

in

an

46), C-protein (41, 53, 55) and other less well studied proteins (55). These proteins are not present along the entire length of the myosin but

actin

structure

antibody-labeling

done

for

proteins

protein

more

be-

of the thin with myosin

M-band

filaments

erably attempt

filaments

summarize

the

ultrastructural

myosin

been

with

I will

about

striated and

19174

the application of these filaments in some detail

cuss

thin

interaction

These

vertebrate

Pennsylvania

sections

learned of the

labeling

the

and

been

of

studying

Although filament

following

has

filaof

Philadelphia,

the

composition

use

structure

them.

composition

In what

fila-

myosin-containing

IMMUNOHISTOCHEMICAL STUDIES”2

of Pennsylvania,

for con-

actin-containing

pp. 543-562, 1975 Printed in USA.

7.

A. PEPE

University

responsible

primarily

FROM

23, No,

surface

in

the along

very (45,

middle the

51). of the

rest

of

544

PEPE

the

length

of the

filament.

due to the presence which are involved myosin

with

rough

surface

is

myosin cross-bridges the interaction of

in

filament

ments

The of

neighboring

the

actin

fila-

creasing 0.2 , are

the clear

almost

always

the filament

(19).

Two packing the

vertebrate

carries

of the

In

one

the

solubility

the

at low ionic portion carries

triphosphatase activity. for the the

characteristics

insoluble globular

(i.e.,

(ATPase)

mole-

strength (0.1)), the adenosine

and

actin

The insoluble rod portion is responsible aggregation of myosin molecules to form

shaft

of

zymatic

the

filament

portion

it is available with neighboring

as

tween

the

act in,

accompanied

portion the

soluble

en-

surface

where

of the

myosin

and

of ATP,

filaments

and

results

shortening

(22). myo(19)

concluded

in the

mid-

from

the

dle

that

of

the

interaction oriented

the

smooth filament

of

rod

the

myosin

molecules,

of the

myosin

increase

in length

of the

by

molecules. one half

results

portion

aggregation occurred

portion

myosin

of and

of

those

in

further leading

filament

aggregation

to

that

molecules at

the

to

both

similarly

Therefore, the myosin of the filament are

oriented

oppositely

ends

myosin

molecules

all

in

oppositely

other

half

filaments

in striated

the M-band,

bridges to the

between middle

of

the

myosin sectional

formed

taken

in

filaments

show

taken

profiles

of the

profiles in

the

immediately

ments

different

In thin filaments tional

clearly

to

can

(48, the and In

le).

the

triangular

the cross-

has

that

in

of the ture

filament

made

up

of

of one

or

The the

struc-

shaft

of the

This

of

the the

will

be enhanced will

cross-

model

predicts

filament, triangular and

be more

as the profile

the

substruc-

easily

detecta-

of the myosin rod as unit is made up of one

myosin

the

substructure

A. If

more

molecule be about

molecule

20

makes

substructure other model

up

each

of

positions

rod

and

of

the

neighboring

“tilted

thickness

would model

will

be observed

in the

myosin

filaments

ingly

more

difficult

to resolve.

substructure

predicts

is

a substructure

As mentioned files are easily tions

taken

band

they

tions

along

c) (48, by

the

increasing nm

the and

profiles

profiles

(Fig.

id).

subunit

structure

sections

were

methods citrate

observing

can

seen (54).

stained

filament

sections 200 A),

with

M-

lb and

shown

that

to as

much

in a 200 kV clear

triangu-

enhancement

In that by

the

(Fig. been

report the

of

the

thick

conventional

of uranyl acetate followed generally used for thin sections

Downloaded from jhc.sagepub.com by guest on March 13, 2015

A.

20

cross-sec-

thickness the

(JEM be

model

to

in thin

previously

section

microscope

lar

this of about

adjacent seen

of the

It has

increas-

conditions

although triangular proeven in thin cross-sec-

clearly

rest

5 1-53).

as

crosssubstruc-

be

Under

resolvable,

immediately not

in

that

and

should

spacing

above, observed

are

predicts a circular

ture

then

A the rods be superim-

380

increases

profile

the In the all in

towards

A.

20

about

molecules this

both

as

than

Th’erefore,

where

myosin unit

axis” (72). The tilt of filament axis in this Taking the diameter

myosin

thicker

the section sectional

one

structural

and around the filament the molecule around the model is about 3 degrees. the

spacing

than

spacing should be greater. the myosin molecules are

equivalent

electron

in-

in

triangular

48).

filament

fila-

On

the

filament.

molecule).

increases

the

ib).

is

so that

(47,

M-band

(Fig.

myosin

an approximately

shaft

for

ble. Taking the diameter 20 A, if each structural

and

rest of the cross-sec-

myosin

cross-sections

of the

of

molecules

(consisting

packed

thickness

rest

proposed

filament units

profile

as 400

cross-sections

along the triangular

be observed

the

cross-

5 1-53). In

M-band hollow

of the

portion

of

in the

filaments

cross-sections taken only occasional profiles

parts

differences

(Fig.

smooth adjacent

have

which

by M-protein

cross-sections taken through myosin filaments have circular sectional

to the other is a narrow

the myosin filaments attaching of the smooth portion. Thin

cross-sections

filament sectional

posed.

myofibrils

extend from one edge of the A-band (Fig. 1). In the middle of the A-band band,

one are

cross-sections

filament. The

than

of

oriented

the

units

the

myosin

muscle

structural

should

From studies of the growth of synthetic sin filaments from myosin solution, Huxley

skeletal

tural

along

been of

model,

section

for interaction Interaction be-

by splitting

between

of the muscle

the

to the

cross-bridges actin filaments.

globular

in sliding

and

is excluded

have

more

even

2).

arrangement

parallel

combining

(Fig.

models

a rod with rod portion

thickness to greater than cross-sectional profiles

seen

(54)

The myosin molecule is essentially a globular region at one end. The cule and

section triangular

by lead (48). It

STRUCTURE

OF MUSCLE

545

FILAMENTS

sarcomere (repeat unit of the striated myofibril); muscle of the fresh water killifish (Fundulus a, longitudinal section. Z, Z-band; I, I-band; A, A-band; H, H-zone; pH, pseudo-H-zone; M, M-band. b, cross-section through the A-band in the region of overlap of thin and thick filaments. The thick filaments have solid circular profiles. Some appear to have triangular profiles. c, cross-section through the A-band in the region of the H-zone (nonoverlap region). Only thick filaments are present. The thick filaments have solid circular profiles. d, cross-section through the A-band in the region of the pseudo-H-zone immediately adjacent to the M-band. The thick filaments have solid triangular profiles. e, cross-section through the A-band 1. The

FIG.

diaphartus).

in the

region

of the

M-bridges. Some I-band. Only thin York.

was

not

certain

penetrated The using holic

this

entire shown

to give thickness

2). Although filaments sections

many profiles ness

thick

staining

thickness in

filament

Figure

is connected

penetration of the the

are

less

procedure

ness

90 nm

than

(Fig.

2b).

On

2 were

stained

triangles with each profile is predicted

throughout legend

to Fig.

profiles of the and solid in

in thickness

increasing

(Fig.

2a),

clearly triangular 250 nm in thickthe

neighboring

section

thick-

cross-sectional

filament parallel 3 are

proposed structural

all of the

profiles

which

apex blunted by the model

model

located

proposed

incompatible

and

by Pepe

with

Squire (72). In studies, Rayns

Downloaded from jhc.sagepub.com by guest on March 13, 2015

by

the

addition, (59) has

the New

filaments appear

as

(Fig. 2c). This for the myosin

(47, 48) in which 12 close packed so that

cated (as shown in the lower of Fig. 2d). These observations the

filaments

almost

by Pepe units are

centrally

thick

f, cross-section through of Marcel Dekker Inc.,

nm

400

have

(see

have about

to about

section.

cross-sectional roughly circular

of the filaments in cross-sections

of its six

of the

of stain

sections

to each

clearly hollow and have circular profiles. Reprinted from Pepe (51) by permission

alcoholic uranyl acetate followed by alcolead citrate under conditions which are

known the

Each

whether

the

sections

M-band.

of the filaments are filaments are present.

9 peripherally right (47,

model

hand strongly 48, 54) proposed

locorner favor and

are by

using freeze fracture observed substructure

546

PEPE

4

4

4

4

4

4

4

4

#{149}.O.O.O.. I O #{149} #{234} #{149} .#{149}. #{149} #{149}4 #{149} i.O #{149} #{149}e#{149}Sd

4

4

4

4

4

4

FIG.

Cross-sections Sections

2.

citrate.’ 90 nm profiles

the

myosin thickness

*

*

filaments

profiles.

both

Whereas

the

muscle

stained with kV on a JEM 200

*

4 *

of the

alcoholic A electron

4

fresh

4,

water

killif’ish (Fundulus followed by alcoholic lead a, cross-sections less than

uranyl acetate microscope.

were obtained at 200 Most of the myosin filaments have solid roughly circular profiles. Occasional triangular b. cross-sections about 250 nm in thickness. Many of the myosin filaments have triangular c, cross-sections about 400 nm in thickness. Most of the myosin filaments have profiles which represent with each apex blunted. d, cross-sectional profiles predicted by the model f’or the myosin filament by Pepe (47. 48). For the filaments in the lower right hand corner the individual subunits in the model are shown. myosin the

filaments

and

substructure

of’ the

collagen collagen

is helically arranged, that in the myosin ments is clearly parallel to the filament Fresh

in

* *

*

were

Micrographs in thickness. can be seen.

triangles proposed filament in

of of varying

4 4

o diaphanus).

4

_____

.1*

tissue

was

f’ixed

in glutaraldehyde

f’ihrils.

thus

consistent

fibrils

Pepe

(47,

filaaxis, followed

Electron

thickness

were ing

out

observed.

staining

represents

repeated

a second

and

third

time.

Using

this procedure the stain penetrates the entire thickness of the sections. (Details of this method are being prepared for publication; F. A. Pepe. G. Tabas and A. Reingold.)

center

the

of obtained

the

act

250

nm

in

the

Information

250 the the

more

ions

less

the

center

the

in

substructure

with

measurements

substructure

electron

Downloaded from jhc.sagepub.com by guest on March 13, 2015

areas

spacing

micrographs

of in

thickness.

of’the

The

to was

observed

bright

is consistent

than

substructure

only

large

A, which A made

2

paint-

filaments

myosin

in-

Figure

after

spacing

filament.

Ofl

sect

only

was

or

nm

in

ion

In

about

filaments

of’

by

of

those

diffract

thickness

of

myosin

spacing

proposed

cross-sections as

filaments. in

spacing

sections

model

by optical

analyzed

about

the

54).

micrographs

creasing

by osmium tetroxide and embedded in Araldite as previously described (48). Sections were picked up on uncoated grids and allowed to dry in air. The grids were then immersed in 10% uranyl acetate solution in methanol for 3 hr followed by four rinses in methanol. They were then immersed for 3 hr in an 0.05% solution of lead citrate (Polysciences Inc.) in 50% ethanol followed by four rinses in 50% ethanol. This cycle of was

with

48,

in

The Figure

3

in the is about

40 of’ 37

of’ cross-sec-

STRUCTURE

tions

of

ments

well

(56).

with

the

preserved

This

in

of

of these

by Pepe

(47,

proper

filaments

getting

an

(69). Using microscopy length

be

There shape

sections a 200 kV thick

been

the

myosin

of

reported. shown side

occurs

FIG. filaments. diffraction substructure

Using that,

some

3.

the

in

microscopy

appropriate of the

the

found,

electron

about smooth

and

different

portion

of’ the

filaments

mus-

tapered

leading

ends

From

these

that

oppositely

present

The

observed

in

it has

consistent

ribbon-shaped

side

filaments

been

(54,

up

80),

bare

it

was

concluded

long

studies

formation and

filament.

The

substructure

spacing

is about

40

A.

Downloaded from jhc.sagepub.com by guest on March 13, 2015

long

central filaments

muscle

(2)

is

of’ synthetic

(54, in length recently

Optical diffraction pattern of the electron micrograph of a cross-section through the The actin filaments were painted out of the electron micrograph shown in Figure 2b and this pattern was obtained. The spacing of the large bright areas represents the spacing in the

are

of’ the

smooth

m more

region.

molecules

no

1.2

with

zone

smooth

to

of’ the

filaments

the

vitro

filament

filaments

reported

in of’

in

filaments

length

that

sections

myosin

long

80)

was muscle

overlap

myosin

seen the

end

entire

f’act be

with

uterine Synthetic

the

middle.

in length

a

(54,

along

filaments.

filaments

without

oriented

all

can

conditions,

to

studies

portion

cylindrical

but

the

(19)

striated

to

to

myosin

myosin

by end

80).

similar

in

with

Uterine

filaments

cylindrihave been been

found

only (54,

muscle

a smooth

in length

rabbit

were

striated

filaments.

the

solution,

observed

that

fOr

observed

had

from

short

the

were

with

evi-

diffrac-

of’ synthetic

from

growth

increased

been

has

obtained

x-ray

studies,

growth

filaments

they

from

filaments

shortest

myosin

portal

the

under no

(67).

filaments

However, in

either

that

contract

microscopic

of

cylindrical

found can

ribbons

myosin

in that

thin

section

muscle

(30, 68) filaments

71)

be

been

muscles

of broad

those

Measure-

from difficulty

in rabbit

filaments

under

form

myosin

controversy

electron

or

The

(2).

aggregation to

within

of

and stereo electron electron microscope,

filaments

ribbon-shaped (61, 69,

tion

uterine

However,

69, 71).

filaments by the

smooth m

has

cle. Both cal-shaped

to

filament

thick with

2.2

(70).

(61,

entire

can

presence

encoun-

identifiable

of these hampered

mesenteric to

(23). model

filaments

cells

be observed

of the

anterior found

muscle

length been

been

myosin

conditions

can

ment of the sections has

thick the

recently when

In studies have

the

smooth

A

35 support

(71). It has dence

meromyosin

547

FILAMENTS

conditions

consistent

48).

visualizing

vertebrate

the

about

difficulties

in

under

light

strongly

Considerable tered

some

strands

findings

proposed

is also

MUSCLE

fila-

myosin

measurement

observation

paracrystals

All

individual

OF

80). have some

myosin optical of the

548

PEPE

filaments

as long

observed.4

This

the length filaments

as 2.3 rm latter

in length

figure

have

been

is consistent

labeled

with

of 2.2 m for vascular smooth recently measured from thick

muscle sections

a

cultured

in

In invertebrate siderable variation of

the

striated in both

myosin

(51).

filaments

the

In

Rodnius

muscle structure in

abdominal

obtained

the

in diameter

of

myosin

solid

tional

profiles

in the

body

hollow where

circular cross-sectional except for a narrow region

of’ the

throughout.

circular

muscles

filament

sectional

of the

hollow

are

observed.

profiles

of the

filament

profiles

with

hollow

along

the

rest

of the

profiles

filaments

of

muscles

probably

myosin

packing

myosin

filaments

of the (4).

in the

both

cross-

flight

vertebrate reflects

available

antigenic

if not

the

of overlap.

of

myosin

region the

preparation

invertebrate

in

all

did along

the

of’ actin

preparations have

obtained

generally

by

produced

antibodies

the proteins associated with the actin but not to actin itself (50, 82). However, tin

was

from

obtained

frog

antiact and sion.

by

skeletal

in was

muscle

antibody

tained isolated actin

(26).

from isolated

Wachsberger results.

into

contaminated

anti-s-actinin Using actin

blasts

injecting

specific This

for

antiactin

mouse from

PR,

fibroblast chicken

Pepe

the

The

for

immunodif’fu-

mouse

actin

was

reacted cells muscle

FA:

fibroalso

with

obactin

as well cells.

Unpublished

(46)

and

the

in may

show

have

of rein

evidence

actin

with helically

of

of

filaments

the

presence

filament,

of

presumably

wound

antigenic the

strands

protein.

striated

in fluorescence

of

Anti-

myofibril

has

microscopy

(43,

44)

as well

(8) and as by x-ray

(63). fluorescent myofibril,

entire

thin

ant itroponin Endo et al. filament

is

is not. Therefore,

labeling

along

the

not

in the

but

antitroponin (43, 44)

labeling showed that

as

formly

It

is present at similar to the (46)

Pepe

availability

the

the

filament

which

by

the

in

in labeling

differences

not

the

microscopy

Z-band

ant itroponin

by from

electron Using striated

antiac-

(16).

studied

diffraction

isolated

rabbits with

as shown isolated

to

filaments

actin

labeling

been

injection

labeling

which

along

is a highly

troponin

Antibody

of

thus

monomers.

Troponin

FILAMENTS

considerably

is consistent

grooves

the

and

in

localization

actin

the

of

sites in the overlap labeling observed

microscopy

in

in

some

myofibrils

periodic

all

filaments

reflect

electron This

A-band.

interaction

differences

differences

tropomyosin

the

observed

may

in can

overlap-

in the

blocking

The

the

who, also

eliminating,

tropomyosin antigenic gion. Antitropomyosin

(46).

of a basic

sites

of

M-band.

(8)

al.

filaments

act in

entirely

(8)

et

(50)

in

region

al.

et

resulted

to

result

overlap

Endo

myofibrils

or the

actin

and

may

striated

filaments

discussed

reducing,

(51).

ACTIN

of the

previously

with

antitropomyosin

myosin

solid

cross-secparts of the

common

the

between

diversity

variations

relationship

portion

with

A-band

muscle

and

the

ping

that

my has

for actin. ant itropomyosin

by Endo

mus-

profiles

the different in different

Z-band

crossin the

filaments

This

of

of the

showed

To

microscopy

I-band

confirmed

label As

muscross-

invertebrate

was

needed.

in electron

the

or label-

of myofibrils

to be specific showed that

labeling

addition,

everymiddle

elongated

filament

and observed

filaments

tional

have

circular region

middle

only

are

labeling

observed

possible Z-band

antiactin

myofibrils

known (46) first

labels This

cross-sectional

structure

myosin

The

been

filaments (13)

of

antiactin

antibody Pepe without

(3, 60). The flight have solid square

in the

not

are

triangular

showing

circular

of filament

insect

profiles in the

have hollow with the narrow

cross-sectional profiles cles of hymenopterans sectional

thick

studies

cross-sec-

arrowworm

where

cles of lepidopterans sectional profiles, middle

The

ing

of

cells.

of the

fluorescent

and

fibrils

it is not

is a part

detailed

knowledge

to 1.5 Mm for are about 200

compared muscle. They

show

the

filaments

(77),

and

muscles

More

the

muscle so far

if actin

cells

in

chicken

results

not.

fibroblast

pattern

embryonic

From

is conlength

different

muscles

prolixus

about 8 Mm long vertebrate striated

there and

mouse

striated

to determine

(2).

A

fibers

produced

distributed

with we

antibody now

entire

Downloaded from jhc.sagepub.com by guest on March 13, 2015

labeled

in the

of the

the thin of

microscopy is not uni-

thin

filaments

of about 400 pattern obtained contained

but

Observation

electron troponin the

of the that

is available

length

Z-band.

prepared know

labeling showed

troponin

along intervals labeling

(8)

against only

A.

but This

is

by Pepe actin, antibody

but

STRUCTURE against filament

the proteins associated with (50). By adding tropomyosin

onin

to purified

crystals,

Hanson

tions

with

the

actin (15)

a spacing

presence

of

striations

striations

and

corresponds

showed

that

with of

normally

associated

filaments

is enhanced

addition

to

the by

to

the about

that

molecule

(7,

43).

These

of

the

kind

that

can

body

labeling

in

of

related

in the

(one

A

shown

the

antibody

troponin

binds

are

an

detailed

be

filain at

the the

elegant

with

Another

component has

been

myofibrils Although

considered the thin involved

yet

clear

in this

of cr-actinin,

It

is

fluorescent may not of the attached

whether

most detailed obtained on vertebrate

These

involve

filament

6S

and

of

C-protein,

using

other

tech-

and

by Nonomura

(40)

Using

absorption

relatively and Ebashi

component

was

Antibody

prepared

of a-actinin

responsible shown

antibody which not cross-react the

lOS

but

not

has

M-band

the

antibody

possible to identify nents of a-actinin or the

filaments.

thick protein

such

other

as

proteins studied

both

than I

fluorescence

any

will

labeling

of the

first and

the

proteolytic

other

consider

then

labeling

myosin

and

the

includes

LMM

molecule

whereas

but it did addition to

soluble myosin

fragment the

LMM

S2

and

to myosin. study the

These A-band

antimyosin

Downloaded from jhc.sagepub.com by guest on March 13, 2015

and and

which

S2 portions has solubility

the in Si

the can

the

intact 0.1

S2 and The myo-

M

KC1,

fragments

are

If precipitated (28) the

rod

includes

both

of the molecule. characteristics

fragments labeling (this

and

HMM

fragments,

conditions. with papain

is obtained

fragment

with

HMM,

rod

The

insoluble

under these is digested

fragment

of the

two

the

(HMM)

portion of the HMM the globular portion.

of myosin are

trypsin

meromyosin myosin.

into rod

like

The by

light meromyosin a portion of

rest

of the

split

molecule

heavy the

portion be

enzymes

28). The represents

representing the the Si representing sin

entire Iyet been

the

extensively in

described

of

rod

further

either the 6S or 105 compowith structural components actin

already

globular

lOS

more

microscopy

action

fragment

by

a-actinin labeled the the Z-band. It has not

fila-

identified.

labeling

and papain (27, (LMM) fragment

been

labeling.

in other

and

proteins.

I have

myosin

Masaki, the 6S

the

analysis

as a rod with a globular region at one end. myosin molecule can be split into fragments

were

component

to

yet been

antibody electron

the is

to be contaminated

labeled the M-band, with actin (74). In

component of band including

Z-band

the

A-

different in the

filaments,

thick protein

antibody

prepared

a-actinin, showed that for Z-band

against

was

antiserum

the

an

the

microscopy.

distinguishable of

impure (32)

such

differences

of myosin

M-band

in electron

components have

have

of

in the

of the

fluorescent

a-actinin

and

cross-reac-

details

as labeling

of

thin filament, to the Z-band

components,

fila-

studies of A-band labeling with antimyosin have been striated muscle fibrils.

a-

antibody labe properly

Two lOS

be

the

muscles

that

labeling

as well

components

anti-

Z-band

or not

differ-

of’ the

obtained

possible

these in

the

structural

The patterns made

using

the

of

patterns

white

cannot

different

comparison

(1,

and

present

studies

from

uncover

red

is similar,

of these

labeling

vertebrate distinct

differences

myosin

involved

of

at

structural

Myosin

myofibril,

in

to be immunochemically

against Endo

labeling

the

attachment.

the

distinguished (32).

by this

a component filaments are

it is not

shown

of localized

structure

smooth

antigenically

red and white antigenically

filaments

myofibrillar

actinin, striated beling.

of the

the

None of

detected

niques.

and

ments. tivity

structural

obtained

conjunction

in of

Even are

and

he

ments.

the actin filament, that the troponin

studies of

to

could

labeling

shown

antigenicity

muscles.

actin

to

myosin in

band

meridional

striated

been

Since

muscle

FILAMENTS

ofvertebrate

(12, 31). muscles

31).

seen

tropomyosin molecules 270 A from one end

example information

11,

cross-

binding to actin of tropomyosin,

showing

distinct striated

ences

with

intervals of 380-390 A along have led to the conclusion binds filament

In

has

385 the

of troponin paracrystals

to

has

to troponin

of troponin)

intensity

Studies and

muscles

antitroponin-C

reflection (63). ments

para-

spacing

normally

components

the

myosin

and which is enhanced by X-ray diffraction analy-

labeled

three

The related

that

MYOSIN

The

cross-striathe

549

FILAMENTS

actin trop-

introduced.

alone

observed.

to

of muscle

of the

that

is therefore

I-band of myofibrils antitroponin labeling. sis

actin

of 380 A were not

the and

forming

tropomyosin

were

these

and

OF MUSCLE

have been used pattern obtained

is discussed

the

The rod similar

below).

to

550

PEPE

Early

studies

labeling tion

of

fluorescent

of myofibrils of myosin

brils

(9,

to the

17).

observed

comere

length

(9, 75,

slightly

strongly

76,

fibrils

the

of’ the

the

weakly

findings

suggested

in the

A-band

with

76)

and

were

both

remain

intact

discussed ing

the below,

this

was

labeling

Pepe labeling

with

length

was

two

in the

middle

of the

bands

actually

in the

made A-band

The

region

middle

the by

of the

slid-

at

bands:

in no specific was

of the

lap

of

thin

pected

A-band

and

from

where

thick

the

sliding

this absorbed bands in the

antibody middle

and

was

A-band

was

to

the

filaments

observed, region

(Fig.

of nonoverlap

4c).

A more

bands over-

would

be

model

(Fig.

4d).

LMM.

HMM

with

myosin

fragments

indication

labeling

of the

The

for

sarcomere

bodies

the

labeling was

obtained

antimyosin of

timyo’sin

was

the

fibrils

A-band

bands indicated

that

specificity by absorbing

with

myosin bands

by

different

used

decreased middle different

these

the

the band

in intensity, of the

in the

(Fig.

A-band

antibodies

either filaments

A-band antiamounts

labeling

of

A-band be

(46,

The

of the at

due

4b)

sites

are

only

where

49))

can

short LMM-

to

because

along of

filaments.

of the

to blocking the

myosin

for

labeling overlap HMM

of fila-

with

the

myosin-myosin

in the structure HMM-specific

is no The

were of the

interaction

or because

available

following labeling

be atributed sites

there

the

nonuniform

interactions involved myosin filament. (b)

actin

to the

in width

the

is

A-band

is restricted

cannot

antigenic

actin

specific

the

labeling of the

increase

edges

results (a)

ment

and

of

labeling.

From

the Some

the lengths

of the

LMM 49).

decreasing for

As

the (46,

four

(46).

decreased

in the

edge

made

when

were

amount

at each

A-band.

absorbed

edge

region

LMM-specific at

this

each

HMM-specific

nonoverlap

and labeling

increase

of the and

of

that

represented of

was

an-

and this was restricted to at all sarcomere lengths

edge

ex-

With at

Therefore, to the

available

study

labeling

antibody

band

the

for

4b).

detailed

antimyosin

of the

of

labeling

A-band

width

(Fig.

of myosin

Absorption

only

of

and

A-band

of re-

specific

concluded.

brightness

this

antibody

in

the

LMM,

antibody

two

in width,

by an

When the

specific for HMM. With only labeling of the two

the

accompanied

with

as for the antibody

specific

was

myofibril.

LMM as well leaving only

muscle contraction the two bands were absent. Disappearance of the two bands in the middle of A-band

an-

myosin

at each

two

filament

the

the

complete

filaments

of

with HMM removed antibody specific as well as the impurities, leaving only

one

decreased

lengths

fragin the

for the removed

limited

middle

These

purified

of the

the

at sarcomere

preby a

present

absorbed

the and

were myosin

Absorption

A-band was observed, the edges of the A-band

in the

to

and

timyosin for HMM

corresponded

decreased,

absorption

digestion.

was

was premyosin LMM

purified

labeling

of

antibodies

The

conventionally

antimyosin

middle edge

work purified

impurities

region in the observed (9, shown that as

length

for

the

to the weakly labeled narrow middle of the A-band previously (75, 76, 78). Furthermore, it was sarcomere

this

55).

myosin.’

antibody impurities

in the at each

contains

53,

trypsin

with

stricted

the antimyosin for sarcomeres

A-band

mm)

resulted the

bands band

in

used

contained

the

and

42,

two the

conventionally

timyosin

further

between

know

(41,

conventional

As is

up of four

of the

A-band.

(15

ments

(46).

(46, 49) showed that pattern in the A-band

rest

short

filaments

in length.

resolved

studies

in

the sliding (22) in

actin

change

discrepancy

model

antibody

and

not

do

from

length

with

now

fragments

pared

migrated

contraction

myosin

and

HMM In

region

myosin

muscle

we

impurities

in length.

in sarcomere

consistent

for

filament

edge

not

model

which

that

which

labeled

increased

change

and was

A-band.

labeled

A-band

antimyosin used using conventionally

length

of the

for

The pared

A-band

a weakly

middle

These (75,

for

in the

contracted

filament

In rest the

except

region middle

78).

of

of the than

sar-

labeling

function

for labeling

of the A-band the A-band.

myofi-

A-band

a

fibrils,

labeled

narrow

the

as

stretched

localiza-

of striated

in

were

the

A-band

Changes

pattern

sible

antimyosin

established

in

of the antigenic the

of myosin labeling

A-band and is corn-

a given

amount at each but did were

of

an-

edge the not.

of two

This

respon-

It has been shown that the impure LMM removes antibody responsible for the stripes observed in electron microscopy (52), which we now know are specific for C-protein, the major impurity in conventional myosin preparations (41, 42, 53, 55). See discussion

of C -protein

Downloaded from jhc.sagepub.com by guest on March 13, 2015

labeling.

STRUCTURE

OF

MUSCLE

551

FILAMENTS

a)

FIG. 4. Fluorescent antimyosin labeling of chicken muscle myofibrils. a, specific antimyosin isolated using column-purified myosin coupled to p-aminobenzyl-cellulose (53, 55). At a sarcomere length of 2.7 four bands are observed in the A-band, two in the middle of the A-band and a narrow band at each edge. At a sarcomere length of 1.8 the bands at the edges of the A-band have increased in width and the two bands in the middle of the A-band are not present. b, anticonventional myosin (see references 46 and 49). The labeling patterns are comparable to those obtained with specific antimyosin at corresponding sarcomere lengths (a). c, anticonventional myosin absorbed with an LMM fragment of myosin obtained by short trypsin digestion of myosin (long LMM) (49). The labeling at the edges of the A-band is absent. Labeling of the two bands in the middle of the A-band is present at a sarcomere length of 2.5 IL. This labeling is reduced in width at a sarcomere length of 2.3 L, and is completely absent at a sarcomere length of 1.7 (where complete overlap of thin and thick filaments occurs). This labeling is HMM (i.e., 51 plus S2-specific). d, anticonventional myosin absorbed with HMM. The labeling of the two bands in the middle of the A-band is absent. The narrow band at each edge of the A-band is labeled at all sarcome’e lengths and does not change in width. This is comparable to the bands at the edges of the A-band observed with specific anti-short LMM in Figure 5d, e and f.

pletely lap

abolished

of thin

and

HMM-specific can be attributed

when thick labeling to

there

filaments in the interaction

is complete (Fig.

4c).

overLack

regions of overlap of the myosin

cross-bridges of

ing

the

antibody sites

(HMM HMM labeling.

are

available

Downloaded from jhc.sagepub.com by guest on March 13, 2015

portion)

antigenic (c) for

with sites

LMM-specific labeling

actin,

mak-

unavailable only

for

antigenic at the

edges

552

PEPE

of the

A-band

rnyosin

(Fig.

affected

by

increase crease

be attributed

taper

of the

modate

of

the

myosin

the

to

the

filaments.

increased

(d)

at each

edge

lengths

was

antigenic

the

below.

can

of the

A-band

at short exposure

of the

and

of other one-

digestion

model. swinging

Also, the idea of myosin cross-bridges out toward the actin filaments to ac-

commodate

the

changes

filaments was idea, movement

in distance

filaments,

on

duced

from

diffraction

x-ray

protein

interaction,

contaminants

DEAE-Sephadex

used

(62),

antimyosin this

as has

been

labeling

in

the

Steiner (29) chromatography

immunogen

and

patterns was also

in the isolated

same above.

myosin purified on DEAE-cellulose

likewise

got

the

same

the

on substrate,

substrate

antimyosin. gave the (Fig.

body to explained

DEAE-Sephadex binding specific

and

eluting

the

The

observations

obtained

by

anticonventional

myosin

other by

specific for same position

obwas

as

those

that

specific

the

antimyosin

(which

contains

proteins) are the the finding that

same the

these other proteins occurs as part of the antimyosin

and

the

the

bound

one

long

shown LMM

the

bands

labeled

comere

length

beled

observed b). How-

obtained

with

served.

A-band

lap

region

length

labeling

at

the

roughly of’ the

decreases

appears of 2.7 m m

the

A-band the

decrease

edges

the

narrow

can be labeling

obtained

in the labeling

myosin

with

described Now

labeling

two

to

A-band

49)

Downloaded from jhc.sagepub.com by guest on March 13, 2015

in the

5a)

the

A-band

is

region

in

to the at sar-

As

middle

of’

to the nonoverthe sarcomere in the and

the

increases

antimyosin

previously (46,

two Sb).

middle

of’

labeling in width

the pattern shown in are the same as those

give

patterns specific

consider

bands

in width,

of’ the

and brightness Figure Sc. These

as (Fig.

(Fig.

and

corresponds A-band. the

distin-

m. This relatively unlais not always clearly ob-

region

Therefore,

the

anti-

clearly

patterns obtained LMM. The labeling

of 2.9 for

of’ 2.7

narrow

produc-

same as those (Fig. 4a and

in width,

except

time

fragment,

The labeling specific for

A-band

length

dif-

5. The patterns

length

increase

papain Two

in Figure labeling

patterns

of

and (53).

for a short

fragment. antibody

c) are the labeling

middle

were

by tryp-

the middle of’ the A-band corresponding space between the two bands observed

the

anti-

prepared

for a longer time (40 mm)

a sarcomere

two

likewise A-band and

(27),

at a sarcomere

totally

an to

were

Consider first the labeling with anti-rod and anti-long in

and

by (28).

a relatively

labeling

fragchro-

myosin

prepared

of myosin

other

LMM

for

short LMM (Fig. Sd, e and are guishable from the other patterns.

At

This chro-

and

myosin

fragments

Sa, b and antimyosin

ever,

de-

d) using

A-SO

was

fragments are and anti-long

(Fig. with

c and

by column

described

producing

the

A-band

p-aminobenzyl-cellulose, was then bound

ing a relatively shorter patterns obtained with

by colas the

A-5O to antibody

specifically

LMM digestion

bands

labeling

to antibody

fragment

mm)

Lowey

55). by

This specific antimyosin same labeling patterns in the

4a).

patterns

The

A-band. Specific antimyosin from antibody prepared using

conventionally purified myosin (53, was done by coupling myosin purified matography insoluble

gel (73).

myosin

discussed

used

shown

patterns

(55) are the work

sin (8

Rod

purified

of precipitated

ferent

4b,

on DEAE-Sephadex

rod

these anti-rod

21).

can be removed the myosin on

column-purified

as immunogen

obtained and umn

of

dodecyl sulfate-polyacrylamide of the purified myosin

when

later de-

(20,

of conventionally

A-50

fluorescent tained

was

studies

purified myosin preparations by column chromatography by sodium electrophoresis

the

introduced. Consistent with this of mass from the myosin to the

actin The

between

were

as previously

The

of LMM

the

(Fig.

of myosin.

coupled specific

explained

49)

in

lathese is dis-

ional myosin has been reinvestiantibody specific for column-pun-

of myosin

then and

protein

between filaments

localization sites

(46,

fragments

eluted

with the sliding filament

restricted

Pepe

matography

sarcomere

anti-C

relationship the myosin

antigenic

anticonvent gated with

third of each half of the A-band. Therefore, as a result of these studies antimyosin labeling was consistently

the by

fied

fluorescent

The and

HMM

ments

labeling

in the middle

Recently scribed

LMM

to accom-

to increased

sites (not LMM)

cussed

The

between

in width

of the due

with detherefore

of bending

distance

Increase

(see

below). proteins

of the

filament

(55)

beling other

filaments)

as a result

pattern

is

filament.

exposure

from

of the

filament

labeling (and

between

out

packing

of the

of the length

to increased

molecules

the

shaft

brightness sarcomere

in distance

portion

where

in the

the

in in

increase

the

4d)

molecules

with

(Fig

4b).

the

labeling

(Fig.

anticonvent patterns

4a)

and ional

obtained

STRUCTURE

FIG.

LMM

5.

Fluorescent

antibody

This

anti-HMM

ant i-S2 was

purified myosin) Column-purified

of myosin.

labeling

a,b and

of

was

obtained

isolated

using

c, specific

MUSCLE

chicken

as follows. HMM

(from

anti-long

muscle

Specific column-

coupled to p-aminobenzyl-cellulose. rod was then coupled to p-amino-

LMM.

553

FILAMENTS

myofibrils

These

specific

labeling

for

patterns

column-purified

rod

and

as those observed with specific anti-rod at comparable sarcomere lengths (see text). At a sarcomere length of 2.9 IL the entire A-band is labeled except f’or a narrow region in the middle of the A-band corresponding to the portion of the myosin filaments where no myosin cross-bridges are present. At a sarcomere length of 2.7 IL four bands are observed in the A-band, two in the middle of the A-band and a narrow band at each edge. At a sarcomere length of 2.0 , the bands at the edges of the A-band have increased in width and the two bands in the middle of the A-band are not present. d, e and f, specific anti-short LMM. At a sarcomere length of 2.9 IL only the narrow bands at the edges of the A-band are labeled. Only occasionally faint labeling in a single narrow band in the middle of the A-band is also observed. At a sarcomere length of 2.7 IL a single narrow band is observed in the middle of the A-band in addition to the narrow bands at the edges of the A-band. At a sarcomere length of 1.9 IL only the narrow bands at the edges of the A-band are observed. g and h, specific anti-S2.6 These labeling patterns are the same as those observed with anti-rod and anti-long LMM at the corresponding sarcomere lengths (see b and c above).

6

fragments

OF

are the same

benzyl-cellulose and this was used anti-S2 from the specific anti-HMM B. W. Drucker, in preparation).

Downloaded from jhc.sagepub.com by guest on March 13, 2015

to isolate specific (F. A. Pepe and

554

PEPE

with anti-short LMM. dle of the A-band at

The labeling a sarcomere

in the midlength of 2.7

Mm is not in the form of two bands (Fig. cannot be correlated with the nonoverlap of the the

A-band

longer

The

since

sarcomere

interpretation

unclear.

If

specific

sites,

it is essentially

absent

length

(Fig.

ofthis it

of 2.9 m labeling

represents the

5e) and region

must

be

middle length

of

LMM-

available

of the of about

in a

at

short

all

sarcomere

sarcomeres

lengths

(l.9ILm

is complete No increase

including

in length)

overlap of the filaments in width of the labeling

Sf). oc-

curred. This is in contrast to anti-rod and antilong LMM where an increase in width of the labeling at the edges of the A-band did occur in fibnils ing

with

short

restricted

sponds 49)

using

(Fig.

From

results

(a)

antigenic

Both

sites

lap

region

the

rest

rod

the

edges

of

are

and

A-band.

for the

short

throughout (b)

LMM

the

fol-

the long LMM share labeled in the nonover-

are

A-band.

most

Antigenic available

(c)

only

Since

difference between rod and the the presence of the S2 portion

of

sites the

at

only

short LMM is of the myosin

ble

throughout In our

ment 4c). long

LMM

specific

the

LMM

fragment

antigenic digestion ment. specific obtained.

must

sites used In

addition fOr

still

which are to produce

the With

to rod S2

have

must

(d)

some

and

LMM,

of myosin was labeling occurs

nonoverlap

region

both LMMfor labeling

and S2-specif’ic sites are at the edges of’ the A-band,

LMM labeling A-band. The

longer frag-

antibody

the

being Sg

be long

S2-specific

throughout

(Fig.

The

removed by the the short LMM

fragment anti-S2,

A-band,

A-band

sites.

brightest

and

h).

in the TherefOre, available although

is restricted to the edges of’ the results are summarized in Fig-

frag-

myosin know

(Fig. that a

with this antibody

myosin

removed

purities

as

and

and

the

labeling

consistent and

(a)

as

with

antibody our labelthis was

of the

specific

for

Si- and S2-specific was specific for LMM

our

recent

findings

the

and

Steiner

(29)

studied

the

labeling

im-

antibody, (Fig. 4d) (Fig.

Sd,

e

f). availability molecule

of different fOr antibody

of’ the

myosin

the

ends

sites of the

molecules

bend

are

further

with

S2-specific

the

sites

of

the

are

loosely shaft

packed of the along

available

except

molecules ment proposed

excluded

to

the

of’ the

LMM

in

model

(47,

Pepe’s

Pepe FA, Drucker

Downloaded from jhc.sagepub.com by guest on March 13, 2015

BW:

can on

(46,

(b)

49).

the a

with portions

surf’ace

on aggregation

and filament

for

region in the middle, consistent both the S2 and Si (i.e., HMM)

at the

presumably

filaments

filament,

to

filament. only

where

actin

are

the

available

filaments

of the

is related

of

more

out

interaction length

filament

characteristics

LMM-specific

tapered

regions of the labeling in differ-

entire narrow having of the

of’ the in the

48).

The

ure 6. Lowey

A-band

findings of Lowey and the HMM preparation absorb anticonventional

antibody

well

of

LMM prepain addition

region

nonoverlap

sites

absorption

confined

structural

of’ the

antigenic

the

antibody

with the (29). Likewise, used to

anti-

LMM

antigenic

Therefore,

to the

6). and

the

we

contains

myosin S2-specific

and

absorbed a long

Therefore, (Fig. 4c);

portions

edges

5

of LMM-

we

LMM-specific

the

the

(Figs.

conventional findings

consistent Steiner previously

defined

LMM-specific

specific for the impurities. ing was specific for 51

ent

at

further

that

with

from recent S2.

was simiconcluded

readily labeled In the recent

have

A-band

fragment

for

sites region

at each edge of the Aantigenic sites are availa-

myosin

of labeling

specific

antigenic

identification

prepared From our

uswork

nonoverlap

in

labeling

conventional

The myosin

to S2

we

the

previous

molecule in the rod fragments, then labeling of the nonoverlap region and the increase in width due

Si

the

rod

are only available and S2-specific

to

the

A-band

sites band

corre-

conclude

above of

previ-

we

can

described

A-band

myosin

to

the rod are of the A-band.

availability

labeling

label-

anticonventional

and

which

of the of

specific

of the

The

4d).

these

lowing.

edges

work

anticonventional ration removed

lengths.

anti-LMM-specific

described

(46,

the

to the

to the

ously

sarcomere

avilable

restricted

HMM-specific

there

(Fig. has

that

mostly

of the A-band. Labeling with anti-rod lan to that described above, and they

the

where

concluded

are

the

filaments 2.7 m.

Note that the labeling at the edges of the A-band is clearly restricted to the edges of the A-band

they

that portions of throughout much

is, at present,

labeling

sites

narrow region in the only at a sarcomere

at Sd).

patterns obtained with antibody prepared ing rod and 51 as immunogens. From their

In preparation.

fila-

shaft unla-

as

STRUCTURE

OF

MUSCLE

555

FILAMENTS

A-BAND S1

I

S2

.

LMM S2

S2

MYOSIN ACTIN

ACTIN

z

z

6. Distribution of myosin-specific antigenic sites available for antibody labeling in the A-band. This diagram is based on the results presented in Figures 4 and 5 and discussed in the text. LMM-specific ant igenic sites are only available at the edges of the A-band where the myosin filaments taper. S2-specific antigenic sites are available throughout the A-band (except for a narrow region in the middle of the A-band where no myosin cross-bridges are present on the myosin filaments). Si-specific antigenic sites are only available in the nonoverlap region of the A-band (except for the narrow region where no myosin cross-bridges are present). FIG.

beled

narrow

ment of

region

corresponds oppositely

therefore, the region

absence of S2. of nonoverlap

(c)

with filaments

(46,

LMM

labeling,

length,

is consistent

on

(46)

and

later

contribute early

48)

studies

protein cussed

proteins

(41, below.

42,

in

pattern

or

reflects

length (S3. clear,

observed

are

considerably observed

the

antimyosin

in electron

oriented

repeat

to which

myosin

A-band of

the

in

7).

The

stripes

are

of

the

are

due

middle middle

of the A-band. one-third or

period

icity

As is discussed of the

C-protein

of myosin binds.

Pepe

mole(47,

added

on

correspond

in

position

previously

seen

with

myosin

cross-

in both it middle

filament

(41.

fluoreshas

been

portion

55).

the

stripes

label-

the The each

in each

Seven

middle

of

the

side

closest

the These (b)

seven

of

other

seven stripes half of the to

these

one-third

and

anticonventional

of

55).

ant i-C-protein to nine

(47, 48, S3, S4) (Fig. 7a). are: (a) specific for C-protein;

Downloaded from jhc.sagepub.com by guest on March 13, 2015

it may precisely

the

A-band,

half

myosin

actin

that

molecules

(41,

located

(Fig.

each

of C-protein.

C-protein

half

a

the

observation

possibility

along

as seven

each

of

studies

microscopy

the

The

in sar-

it remained

end

microscopy

is observed are

7b).

discussed.

In electron of

that

spe-

primary contaminant of myoconventional methods (73).

electron of the

(Fig. change

reflecting

different

peniodicity the

to be present

stripes

55)

of this

the

antibody-labeling

half

was with spe-

the

was

shown

mi-

of

A-band not with

a way

and

orientation

half

the

with

the but

antibody

C-

4),

that

stripe

meaning

in

anticonventional the

(Fig.

The

C-protein is the purified by

ing

the

specific

periodicity

with

from

although

represent

each

the

of

a single

(53,

in such

distance

and

diswere

in

observed

half of myosin

in position

cence

using either anticonven-

stripes

below,

be

was

myosin

changed

From

particular

to

assumption repeat

However,

for

antibody-labeling

specific

patterns

with

in

immunogen specific

the

labeling

antirabbit

comere

sin

the

spite

is due

the

in each antichicken

stripe

in

in

periodicity

superimposed

prepared

which will no differences

fluorescent

to specific labeling

cules

and

of the A-band

labeling

in detail

may

labeling

as the antibody

53, 55), Although

the

myosin

A-band

observed specific

is not

of S2-specific

antibody

conventional myosin and thus contained

observed

croscopy

periodic

filaments

hinge

(49)

of antimyosin involved

A-band

seven

out

exposure

microscopy

other

the

swings

the

the reflects

constant

diffraction

of

Therefore,

antimyosin.

cific

studies

by x-ray

bridge

myo-

reflected

molecules

is valid.

No

filament first pro-

stripes

myosin

of C-protein,

bridge

using

tional

the myosin This was

distortion

to increased

electron (47,

the

labeling

cific

sarcomere of the

that

peniodicity

of

these

of

filament.

finding

in bright-

in

that

periodicity

myosin

for broadening A-band) and

swinging

substantiated

in is

sites

of antibody-labeling

addition,

when

sites. The

Increase

assumed

myosin

overlapping

decrease with

basis

In

region

(d)

and,

antigenic

with

away from with actin.

the

studies.

Si

48)

repeat

labeling only the filaments

(accounting edges of the with

sin cross-bridges on interaction

Sl

of

49).

fila-

overlap

molecules

interact

labeling at the

of the

of LMM

of

blocking

bridges

ness of S2 of labeling

posed

middle

region myosin

the

actin

the

oriented

consistent when

in to the

two to

the

in the A-band stripes myosin

seven stripes reflecting the

556

PEPE

FIG. 7. Antimyosin labeling of chicken muscle myofibrils in electron microscopy. a, anticonventional myosin. The seven stripes in the middle one-third of each half of the A-band are due to labeling of C-protein (41, 53, 55). The C-protein is present at intervals determined by the myosin molecules to which it binds (55, 64). b, anticolumn-purified myosin. Labeling is specific for myosin. The single stripe in each half of the A-band varies in position

repeat which 4:30

with

periodicity

A

the

eighth

(41,

55).

The

in

at

one.

the other

heavier

fiber only

In

to of

where seven

some

anti-C-protein labeled

Taking

experiments coupled

the

strongly

eighth

stripe

rethe

same

C-

one that

weakly

or not

at

to specific

cent seven and

to the bands

than

in

width

labeling

of

was

overlap

of

the

labeling

concentrated

Downloaded from jhc.sagepub.com by guest on March 13, 2015

in the each

shortened

of the

present in

on

not

the

two side

fluores-

location

to

the

microscopy, fluorescent

corresponded

eighth and ninth labeling of the noticeably

filaments.

the

The

in electron width of the sarcomeres

A-band

of

sarcomeres,

A-band.

corresponded

in stretched

ant i-C -protein

bands

being

middle

stripes observed the additional

stripes

the

as two sarcomeres

wider

additional

ninth

the

due

microscopy

the position of the electron microscopy,

labeled from

are

stretched is

bands

anti-C-protein of antiserum.

from and

bands the

it was labeled of each

and that the eighth and ninth stripes labeling of two other as yet unidenti-

is observed In

closest

to p-aminobenzyl-

obtained

stripe

A-hand

together

the consistently middle one-third

antigens.

(55).

fibnils

the

of’ the

labeling

C-protein fOr

observations

In fluorescence

labeled

except

these

C-protein represent fied

presumably stripes are the

stripes

was used to isolate different preparations

eighth

all.

concluded that only seven stripes in the

clearly

labeling

with

labeling all

variable are

a fiber

anti-C-protein

preparation

cellulose from two

of

presumably

absence

are

stripes

fibrils

prior

the

protein

to

about (41, 47,

C-protein are labeling,

stripes ninth

in the occurs

equivalence in

ninth and

where

Mixing

ninth

The

and eighth

deeper labeling

labeled.

molecules

half

peripheral

anti-C

occurs; lighter

myosin

(53).

is bound; (c) spaced (d) about 150 A wide

stripes specific for seen with anti-C-protein

hut

with

the

length

64).

The seven consistently

labeled

in sarcomere

of

the C-protein apart; and

48, 53-55,

sults

change

diminished

However, in narrow

to

stripes. eighth

stripes

In and by

antibody is more

STRUCTURE easily

detectable

in electron

fluorescence

microscopy

conceivable

that

ninth

stripes

but of the

antibody

labeling

the

eighth

and

ent

from

C-protein

seven

These

ninth

eighth

and

the

greater

in

ponent and

reduction

stripe

antigens

are

is responsible

emphasize was

studies

that

were

present

from

tion

differ-

components and

in

copy

indicated

were

present,

beled

the

have

the

fluorescence

against

labeling

and

electron

micros-

contaminating

particularly and

of

antibodies

antibodies ninth

Turner, shown

patterns

which

stripes

with

to

myosin

la-

varying

intensity.

Since

C-protein

ments is

binding

is determined

bound

(54,

involved

by the 55,

in any

determination likely

the

myosin

protein in both

croscopy. Using beled both the absorbing sin, Pepe different

M-band

and

Ebashi

(21).

length

It is

determimyosin

these

studied and

which I-band,

laand

and

isolated

absorb

from and

Samosudova

micrographs

that the

be

a absorb Masaki M-band

polypeptide

the

extracted

protein

the

M-band

labeling

extracted

the

component antibody and

Takaiti

protein chain

to

into weights

entire

To

sary

identify that

and

structural

made.

From

structural

M-band

or 860

A

can

have

(48),

from

M-band

been

been

measured

been

equally

on each

48,

52,

(24,

the M-band, myosin

for

and

Carlsen

also

the

where

transverse

at each

(33)

further

re-

dinal

sections.

two

components

BL,

the

Downloaded from jhc.sagepub.com by guest on March 13, 2015

52, six

filament, myosin

Pepe

proposed

along are observed

addition,

Mochan PK,

M-band (24), there

position stripes In

the

53).

model

labeled

9Chowrashi

along

each of the six neighboring (24,

8Eaton

as 750

filament

bridges

the

of up to five

spaced

myosin

In

A-band,

it consists

including

the

has proposed.

has

53). In cross-sections

filaments

reconstitute

in fibrils removed.

the

of the

are observed

be neces-

through and

stripes portion

compo-

it will

of the

of the

which

and

been

protein

models

A

which

of 165,000

the

width

bridges

weight

phosphoryl-

of the

not

the M-band has been

sections

Knappeis

could

has

unequivocally

longitudinal

In

have

chain

it as the

do this

one between

A-band

has

ultrastructure

middle

have form

and Pepe (5) does have activity. Chowrashi and found that the 100,000-

M-band

to show

(24)

(79) muscle

Mochan8

component

the structure of which the M-band The

which

obtained so far it is clear that of a protein in the M-band is not

transverse

extracted

antibody which labeled both the M-band. Masaki, Takaiti

(3S)

the results localization

and

40,000-dalton

identification

of the

which

I-band. Morian M-band

of 40,000,

Eaton

the

weight

described

and tropomyoM-band protein

could

that

protein

enough

by antibody electron mi-

proteins.

muscle

showed

chain

nent.

antibody and the

electron

striated

specifically

with

fluorescent M-band

specifically

the

length

by 5 mM Tris buffer, pH 8. Kundrat (25) used this procedure to extract the

antibodies the I-band

solved

be

for

is in the

has been fluorescence

from

and

M-band.

cannot

weight

kinase.

activity. At present

labeling

of the two components and Pepe (5). Recently

component of Eaton some creatine kinase Pepe9 have recently ase

specifically

M-band

and Eppenberger component is the

that

dalton it

(48).

from

of

completely and Pepe M-band

for

filament

fila-

to which

filament

it with actin, myosin (46) showed that the

(65) showed

could

myosin

themselves

M-band labeling

was

mechanism

information

of the

molecules

C-protein

vernier of the

that

nation

64),

the

myosin

creatine

confirmed

these

preparations

a chain

to one by Eaton

and

against

M-band

antibody

Walliman that this

components

of 100,000

prepared the

the

for extrac-

two

both the M-band and the and Harrington (36) isolated with

Eaton of

(25)

absorbed

from

corresponds obtained

anti-C-protein

Antibody

specifically

protein

anti-

obtained

However,

eighth

immu-

contaminating

antiserum

that

to be interfrom

specific

labeled moto

Pepe

weights

labeled

also

(34).

identified chain

is corn-

modification

and

M-band,

con-

94,000-dalton

a

of Kundrat

daltons.

have

component

the

using

polypeptide

40,000

they

phosphorylase

(5),

of the

with

which

and

is probably Pepe

for the with

obtained

in

the

(42).

observed

care

experiments

evidence

nodiffusion

C-protein

the

recently

165,000-dalton

protein

procedure

corresponding

more

M-band

by

557

the

antibodies

labeling No

isolated

that

stripes.

antibody

bodies

daltons;

cluded

stripes, by filament the conclusion that

which

studies

preted.

94,000

microscopy

The

ninth with

in

detectability

in electron

eighth and is consistent

other

below

not

filaments.

of fluorescent to the overlap,

of the

FILAMENTS

it is

than

SO); therefore,

labeling

is reduced

fluorescence overlap

microscopy (48,

the

OF MUSCLE

BS: FA:

by

are six Mthe

filament in longitu-

M-filaments

are

Unpublished

results.

Unpublished

results.

B

P L

b)

A N E

C Model for the M-band of striated myofibrils. a, three-dimensional representation of the attachment of M-bridges between neighboring myosin filaments. In longitudinal sections a maximum of five stripes are observed in the M-band (24, 52, 53). Only the M-bridges contributing to the middle three of these stripes are shown. The myosin filaments are numbered. The bridges labeled (a) are all at the same level and are all parallel to each other (similarly for the bridges labeled (b) and (c)). b, representation of the model viewed from the top. This represents a cross-section through the M-band. Each myosin filament has six M-bridges, one to each of the FIG.

neighboring

8.

six

myosin

filaments.

The

three

lines

labeled

plane

A, plane

B and

plane

C indicate

different planes labeled (a),

of section all parallel to the long axis of the myosin filaments. Plane A is parallel to the M-bridges plane B is perpendicular to the M-bridges labeled (c) and plane C is perpendicular to the M-bridges The pattern of stripes expected in the M-band in these three different planes parallel to the myosin shown in Figure 9a. 558

Downloaded from jhc.sagepub.com by guest on March 13, 2015

labeled filaments

(b). is

STRUCTURE

present

in the

myosin

filaments

occurring

ment.

at

successive

model,

position

section

taken

should

give

stripes

in

The

that

positions

the

filament,

the

same

53)

was

stripes

the

M-band

based

patterns

on

described

(52, the (b)

his

to

model of

M-band and

in

These

PLANE

later in the

three

of (a) zone stripes

(d)

concerning and

the

a neighboring

five

The

three

one

of the with

The

the

other

two

M-band.

consistent the his of

between used

this

model

model,

middle of levels

the

M-bridges

B

the

M-band 48)

published

M-bridges

at

in parallel.

are

in by

(47, filament.

be

60

PLANE

abc

C

for the

assumptions

myosin

is changed

in

middle

model

will

positions

the

shown the

all of the

three

middle more

by Pepe

the

of

outer

make

the

filament for

The clearly

This

with

model

the

model

relation

In this

direction

the

(c) of the

of two

which

considered

myosin

of the

stripes. were

only

of the

successive

PLANE

pair

I have

details

elsewhere.

three present

three, side

(e) only

In

in deriving

were

middle to one

(52).

is

M-

of the added

of’ five

stripes

out

compli-

studies

consisted

8,

M-band

M-band

side being

three stripes, and

than

Figure

the

the

formed

A

b

for

pointed

middle of the bare the maximum of five

a

three,

distinct

five

antibody-labeling it was

of’ stripes 53).

one

two of the middle three stripes when

by Pepe

observations

section (48),

patterns in

of’

fibril

three

proposed

from

in the

different stripes filament,

middle

on each

the middle

plane the

of

stripes,

fila-

bridges

any

through

added

four

maximum

in longitudinal

cated

six

559

FILAMENTS

one

M-band. for

the

to the the

are

pattern

MUSCLE

M-bridges

along

there

along

the

parallel the

since

filament derived In preliminary

band

are

connect

longitudinally

model 52,

myosin studies.

These

and

In this

at each

(48,

M-band.

OF

the

three

degrees.

A

C

abc

1

a)

2 3

b)

FIG.

molly

9. Pattern

(Mollienesia

of stripes

observed

in longitudinal

sections

through

sphenops) and the corresponding patterns predicted by the model for the planes A, B and C indicated

stripes predicted M-bridges at position a. The M-bridges plane A. All planes of’ longitudinal section

Plane B, perpendicular the shaft of the myosin

at

positions parallel

a, b and c will all to any of the M-bridges

the

M-band

by the model

of the

muscles

in Figure

of the

black

8. a, pattern

of

in Figure 8h. Plane A, parallel to the be observed in a longitudinal section in will give three stripes in the M-hand.

to the M-bridges at position c. The M-bridges at position c will all be superimposed on filaments and will not contribute to stripes in the M-band; thus, only two neighboring

stripes will be observed. Likewise, a plane will give only two neighboring stripes in the M-bridges at position b will be superimposed

stripes in the M-band; thus, only the outer patterns of stripes observed in longitudinal predicted in (a) for the three diff’erent planes

of longitudinal M-band. Plane on the shaft

section perpendicular to the M-bridges at position a C,. perpendicular to the M-bridges at position b. The of the myosin filaments and will not contribute to

two of the middle three stripes will be observed in the M-band. h, sections through the M-band (52, 53) corresponding to those of longitudinal section through the model for the M-band in Figure

8.

Downloaded from jhc.sagepub.com by guest on March 13, 2015

560

PEPE

longitudinal cated

section

in

M-bridges

8b

will

Likewise, (c)

are angled Any plane parallel stripes.

(a).

a set

of M-bridges

A

longitudinal

the

shaft

of

the

labeled since

they

of section. which is

will

section

M-

show

taken

in

8b

is perpendicular

labeled

(c).

Therefore,

(c)

will

filaments

the 13.

on the

and

12.

all

be superimposed

myosin

9. Finck H, Holtzer H, Marshall JM: An immunochemical study of the distribution of myosin in glycerol extracted muscle. J Biophys Biochem Cytol 2:175, i956 10. Goldman M: Fluorescent Antibody Methods. Academic Press, New York, 1968 ii. Gr#{246}schel-Stewart U: Immunological evidence for human myosin isoenzymes. Immunology i7:99i, 1969

three

in Figure

M-bridges labeled

of

M-band.

bridges

to stripes

respect to the plane longitudinal section

to

set

these in the

of the

contribute

with of

A mdi-

the

Therefore,

projection

will

plane to

to stripes

B indicated

bridges

in the parallel

contribute the

(b) and

to

is

labeled

bridges

plane

taken

Figure

will

i4.

not

contribute to stripes in the M-band. In plane only two of the three stripes will be observed

B in

is.

the M-band. These will be in the neighboring positions corresponding to the position of the M-bridges

labeled

section taken is perpendicular This

will give two

missing. are

(a) and

These

shown

in

patterns through

stripes with

patterns

longitudinal

9a

and

in

M-band

This variation

in Figure 8b labeled (b).

the middle

predicted

Figure

observed the

(b). A

in plane C indicated to the M-bridges

by the

the

are shown

18.

sections

in Figure

9b.

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