Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1991 Selected properties of the cytoskeletal and its interactions with at the muscle Z-line Stephan Roman Bilak Iowa State University

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Selected properties of the cytoskeletal protein synemin and its interactions with proteins at the muscle Z-line

BilaJc, Stephan Roman, Ph.D.

Iowa State University, 1991

UMI 300N.ZeebRd. Ann Aibor, MI 48106

Selected properties of the cytoskeletal protein synemin

and its interactions with proteins at the muscle Z-line

by

Stephan Roman Bilak

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Animal Science Major: Muscle Biology

Approved: Members of the Committee:

Signature was redacted for privacy.

In Charge of Major Work

Signature was redacted for privacy. Signature was redacted for privacy. theMajor Depé^ent

Signature was redacted for privacy.

For the Graduate College

Iowa State University Ames, Iowa

1991 ii TABLE OF CONTENTS

Page

GENERAL INTRODUCTION 1

SECTION I. SELECTED PROPERTIES OF MUSCLE SYNEMIN AND IDENTIFICATION OF A MAMMALIAN HOMOLOG 39

SUMMARY 41

INTRODUCTION 43

MATERIALS AND METHODS 46

RESULTS 58

DISCUSSION 103

ACKNOWLEDGMENTS 113

REFERENCES 114

SECTION II. INTERACTION OF PURIFIED SYNEMIN WITH MUSCLE Z-LINE PROTEINS 121

ABSTRACT 123

INTRODUCTION 125

MATERIALS AND METHODS 128

RESULTS 136

DISCUSSION 151

ACKNOWLEDGMENTS 157

REFERENCES 158 ill OVERALL SUMMARY 165

LITERATURE CITED 169

ACKNOWLEDGMENTS 195 1 GENERAL INTRODUCTION

Vertebrate Structure and Major Properties of the Myofibrillar Proteins

Tliree types of muscle are recognized in vertebrates, including skeletal

(striated and voluntary), cardiac (striated and involuntary), and smooth

(nonstriated and involuntary) muscles. The fundamental structural unit of skeletal muscle is the individual muscle fiber or cell [for reviews, see Goll et al.,

1984; Huxley, 1988]. The cytoplasm () of the multinucleated fiber contains the usual insoluble complement of subcellular constituents such as the Golgi apparatus, mitochondria, ribosomes, glycogen granules, and endoplasmic (sarcoplasmic) reticulum. When the muscle cell is viewed with the light microscope, alternating dark (A-band) and light (l-band) bands are observed [Huxley, 1953, 1957, 1988; Huxley and Nigerderke, 1954].

This banding pattern is the result of the presence of a highly organized array of cylindrical protein threads (1-2 //m in diameter), called , which are oriented in register and parallel to each other and which extend the length of the muscle fiber.

Electron microscopy conducted on thin sections of striated muscle reveal that the A-band and l-band pattern results from two interdigitating filamentous systems, namely the thick (-containing) and thin (-containing) filaments, which alternate and produce the characteristic striations observed along the [Huxley, 1957, 1963, 1988]. The H-zone is a less electron- dense region in the center of the A band of relaxed myofibrils where thin filaments are absent. A narrow, electron-dense M-line bisects the A-band

[Knappeis and Carlsen, 1968; Luther and Squire, 1978]. Each i-band is bisected by a very electron-dense Z-line structure. The is considered the basic structural unit of the myofibril and the physiological unit of muscular contraction, and is defined by the distance (-2.3-2.8 //m in resting muscle) from one Z-line to the next Z-line. The thin filaments are anchored at the Z-line at one end, where they form a square lattice in cross section, and are present throughout the I-band. The free ends of the thin filaments extend some distance (to the edge of the H-zone) into the A-band, where they are arranged in a hexagonal array in cross section [Huxley, 1957].

Thick filaments (~15 nm in diameter) are composed primarily of myosin, which is a long (-160 nm), mostly rod-shaped (-150 nm) molecule with double "heads" or globular projections (-15-20 nm long) at one end [for a review, see Squire, 1986].

. Myosin is a hexamer (three pairs of subunits), containing two major (heavy) polypeptide chains (-200 kD each) and four light chains (-20 kD each). The heavy chains form all of the long myosin rod (a coiled-coil structure) and most of the myosin head region. One each of two pairs of light chains bind to each of the globular heads [Kendrick-Jones et al., 1976; Squire, 1986]. Two important properties of myosin are that it is an adenosine triphosphatase

(ATPase), and that it interacts with actin during [Tokunaga et al., 1987]. Proteolytic fragmentation studies have shown that each myosin

head region contains both ATPase activity and the actin-binding site [Vibert and

Cohen, 1988]. Myosin molecules will spontaneously assemble into thick

filaments in vitro, first by packing in a tail to tail (antiparallel) fashion and,

secondly, in a head to tail (parallel) fashion as the filament elongates [Offer,

1974]. Presumably, this process occurs in vivo, in such a way that the rod portions of myosin molecules form the cylindrical backbone of the thick filament and the molecules in one half of a thick filament have the opposite orientation

(polarity) to those in the other half. As a result, the central region of each thick filament (bare zone or pseudo H-zone) is free of the myosin head projections, which project from the thick filament surface at defined, ~43 nm intervals

[Huxley, 1963; Offer, 1974; Squire, 1986].

C-protein (~140 kD) [Offer et al., 1973; Starr and Offer, 1978], initially identified in myosin preparations by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [Starr and Offer, 1971], has been shown to bind to myosin. In the muscle thick filament, C-protein is found in the cross bridge region in each half of the bipolar thick filament in seven to nine evenly-spaced bands (43 nm apart) [Offer et al., 1973]. The physiological role of C-protein remains unknown, although it has been suggested that it may function to stabilize thick filament structure during contraction and/or have a role in control of thick filament assembly [Offer et al., 1973; Starr and Offer, 1978; Koretz,

1979]. It also has been suggested [Yamamoto and Moos, 1983] that C-protein 4 may play a role in contraction because it affects actomyosin ATPase activity

[Offer et al., 1973] and can bind directly to F-actin [Moos et al., 1978].

Recent, primary structural studies (from cDNA derived sequence) have shown that C-protein belongs to the immunoglobulin superfamily [Einheber and

Fischman, 1990].

Thick filaments are held in register at their centers by M-line structures

[Knappeis and Carlsen, 1968; Carlsson and Thornell, 1987]. Three to five sets

(when viewed longitudinally; depending on the muscle fiber type and/or species)

[Pepe, 1983] of transverse M-bridges connect each thick filament in a hexagonal arrangement to six neighboring thick filaments, which can be seen in cross section through the M-line region [Knappeis and Carlsen, 1968; Trinick and Lowey, 1977]. Thin M-filaments, running parallel to the myofibril axis, are thought to interconnect the transverse M-bridges at their midpoints [Luther and

Squire, 1978]. In addition, Y-shaped secondary M-bridges are thought to connect adjacent M-filaments in transverse register [Luther and Squire, 1978;

Carlsson and Thornell, 1987]. Myomesin (-185 kD) [Grove et al., 1984,

1985] and muscle specific creatine kinase (~84 kD, consisting of two ~42 kD homodimers) [Turner et al., 1973; Wallimann et al., 1983] are thought to make up at least some of the M-bridges. Another protein considered an M-line constituent is M-protein (~165 kD) [Trinick and Lowey, 1977], and it may comprise the M-filaments [Woodhead and Lowey, 1982]. The exact function of the M-line proteins remains uncertain because in vitro studies have failed to 5 demonstrate significant interaction among M-iine proteins and myosin

[Woodhead and Lowey, 1983].

Two liigh molecular weight (220,000 and 200,000) polypeptides, first recognized by SDS-PAGE analysis of actomyosin-extracted (cytoskeletal- enriched) fractions of bovine [Price, 1984], were later shown by immunofluorescence microscopy (using antibodies prepared against the cardiac proteins) to be located at the periphery of the mammalian skeletal muscle myofibrillar M-line and named skeiemins [Price, 1987]. These two proteins, which are highly homologous to each other as demonstrated by peptide mapping, also are present in mammalian [Price, 1984]. The immunofluorescence labeling studies showed that antibodies directed against bovine cardiac skeiemins label the M-lines of mammalian cardiac and skeletal muscle in a continuous pattern (~4 nm wide) and, when viewed in cross section, indicated that labeling was present at the periphery of the myofibrillar

M-lines [Price, 1987]. The antibodies to mammalian skeiemins did not cross- react with any chicken muscle proteins [Price, 1987]. The exact role(s) of the skeiemins remains unclear, but skeiemins may comprise filaments that run transversely to the myofibril axis at the M-line level, and link adjacent myofibrils at this position. It has been suggested that the skelemin filaments may provide additional structural stability for the myofibrils during contraction/relaxation cycles [Price, 1987].

The thin filaments of striated muscle are approximately 1 //m in length by 6- 8 nm in diameter, and contain a backbone formed by a two-stranded, helical

polymer of actin [for a review, see Payne and Rudnick, 1989]. In solutions of

very low ionic strength, actin exists as a globular monomer (G-actin) with a

molecular mass of ~42 kD [for a review, see Pollard and Cooper, 1986]. It has

been shown that globular actin is bi-lobed in appearance, having a large and a

small domain separated by a narrow cleft [Kabsch et al., 1990]. Actin

monomers bind both adenosine nucleotides and divalent cations [Poliard and

Cooper, 1986; Kabsch et al., 1990]. As the ionic strength is increased to

approximately the physiological level by addition of KCI, G-actin polymerizes

into a twisted, double strand of actin beads (F-actin) [Holmes et al., 1990].

Because the actin monomers are asymmetric, and because assembly occurs in a

head to tail fashion [Pollard and Cooper, 1986], the F-actin filament is polar.

During actin filament assembly, the bound ATP is hydrolyzed to ADP, with

release of organic phosphate [Carlier and Pantaloni, 1988]. The polar actin

filament has a diameter of about 7 nm and contains structural repeats about every 36 nm along its long axis [Holmes et al., 1990; Valentin-Ranc and Carlier,

1991].

Two important regulatory proteins, and , associate with actin filaments [for a review, see Payne and Rudnick, 1989]. Tropomyosin is a long (-40 nm) slender molecule, and is comprised of two, ~ 100 % alpha- helical subunits that form a two-stranded, coiled-coil dimeric rod. Tropomyosin molecules are joined in a head to tail fashion and run the length of the thin 7 filament in each of the two grooves formed by the two intertwining F-actin

strands [Moore et a!., 1970; Trombitas et al., 1989, 1990]. Within each groove, each tropomyosin molecule is in close contact with seven actin monomers, and imparts cooperative communication among actin monomers of the thin filament [Hitchcock-DeGregori and Varnell, 1990]. A troponin molecule consists of three unique polypeptide chains, troponin-C (TN-C), troponin-l (TN-

I), and troponin-T (TN-T) [Greaser et al., 1972], and binds near the C-terminus of each tropomyosin molecule located along the thin filament. The troponin complex (molecule) functions as a Ca^^ sensitive switch in the regulation of muscle contraction [Ebashi and Endo, 1968]. TN-T (~31 kD) binds to TN-C and TN-I and binds the entire troponin molecule to tropomyosin. TN-I (~21 kD) functions to inhibit the actin-activated myosin ATPase activity in the resting state [Payne and Rudnick, 1989]. TN-C (~18 kD) [Potter and Gergely, 1975] reversibly binds Ca^^ ions and is responsible for removing the inhibitory effect imparted by TN-I on the interaction of actin with the myosin cross bridges [Zot and Potter, 1987]. The release of calcium (~ 1 //M) from the initiates a series of conformational changes among the troponin subunits, ultimately leading to movement of tropomyosin, which in turn allows the myosin cross bridges to make contact with actin during muscle contraction

[Zot and Potter, 1987]. As the calcium concentration decreases (<0.1 //M), this series of events are reversed, tropomyosin again exerts its constraints, and the muscle relaxes. The thin filaments are attached and held in square register at one end by the

Z-line structures [Huxley, 1954; Knappeis and Carlsen, 1962]. All of the thin

filaments on one side of the Z-line are anchored with the same orientation,

whereas those on the opposite side of the same Z-line have the reverse

orientation [Huxley, 1963]. Thus, the polarity is reversed on either side of the

Z-line. A cross section through the Z-line shows that the thin filaments are held

in a highly ordered square lattice array comprised of Z-filaments [Yamaguchi et

al., 1985; Goldstein et al., 1990; Luther, 1991]. The Z-filaments are thought

to be comprised of the protein a- [Yamaguchi et al., 1985].

ff-Actinin was discovered by Ebashi et al. [1964], and later shown to be located

at the Z-line of skeletal muscle [Masaki et al., 1967; Robson et al., 1970;

Stromer and Goll, 1972; Schollmeyer et al., 1976]. The a-actinin molecule

( — 200 kD) contains two subunits ( — 100 kD each) [Suzuki et al., 1976; Endo

and Masaki, 1982], which are arranged in an antiparallel orientation [Arakawa

et al., 1985; Wallraff et al., 1986; Arimura et al., 1988]. Each end of the a-

actinin dimeric molecule has an actin binding site. This site is present in an

~36 kD amino-terminal domain of each subunit [Imamura et al., 1988]. Two

EF-hand-like sequences (similar to the four divalent cation binding sites in TN-C) are found in the carboxyl-terminal region of each subunit [Baron et al., 1987].

The central rod domain, which is responsible for the anti-parallel arrangement of the overall a-actinin molecule, is composed of four -like repeating units

[Baron et al., 1987; Arimura et al., 1988]. Although each a-actinin molecule 9 presumably binds to two thin filaments in the Z-line [Yamaguchi et al., 1985],

the exact nature of the F-actin/a-actinin interaction remains poorly understood

[Goll et al., 1991].

While actin (those molecules inserted into the Z-line domain) and a-actinin

are considered the major proteins of the Z-line structure, a number of other

proteins, often present in small amounts, also are located at the Z-line. In many

cases these proteins are poorly characterized, and can be divided into those

that are located at the outer edge or periphery of the myofibrillar Z-line

(peripheral Z-line proteins) and those proteins that are located inside the Z-line

where thin filaments are inserted (integral Z-line proteins like o-actinin).

Peripheral Z-line proteins include [Lazarides and Hubbard, 1976;

Richardson et al., 1981] and synemin [Granger and Lazarides, 1980; Sandoval

et al., 1983], which will be described in more detail in the next section.

Another protein located at the periphery of skeletal [Gomer and Lazarides,

1981] and cardiac [Koteliansky et al., 1986] myofibrillar Z-lines is , a high molecular mass (2 subunits of ~250 kD each) actin-binding protein that also exists in non-muscle cells [Wang et al., 1975]. Each filamin subunit has one actin binding site [Weihing, 1985]. The two filamin subunits are attached at one end of the long ('-160-190 nm) molecule [Gorlin et al., 1990] and, at least in vitro, can cross link actin filaments into three-dimençional networks

[Weihing, 1985]. Filamin's role in striated muscle cells is unclear. Small amounts of spectrin [Nelson and Lazarides, 1983] and (goblin) [Nelson 10 and Lazarides, 1984] also have been shown by indirect immunofluorescence to

be concentrated at distinct foci along the adjacent to the Z-line,

and at the Z-line periphery, when striated muscle is examined in cross section.

It has been suggested that these proteins may serve as anchorage sites for the

intermediate filaments (IPs) at the plasma membrane [Georgatos and Blobel,

1987a]. In vitro studies, which have demonstrated interactions between

desmin and spectrin [Langley and Cohen, 1986] and between desmin and

ankynn [Georgatos and Marchesi, 1985], add support for this suggestion.

A recently discovered protein that has generated considerable enthusiasm is

Cap Z [Casella et al., 1986, 1987]. This integral Z-line protein is a

heterodimeric protein consisting of two subunits (~33 and ~31 kD) [Caldwell

et al., 1989; Casella et al., 1989] and selectively binds to the barbed-end of an

actin filament in vitro [Casella et al., 1986, 1987]. Immunoelectron microscopy

studies on frozen sections of chicken skeletal muscle showed that this protein

is distributed throughout the Z-line [Casella et al., 1987]. Four poorly

characterized, integral Z-line proteins include eu-actinin {~42 kD) [Kuroda et al.,

1981], Z-protein (~55 kD) [Ohashi and Maruyama, 1989], zeugmatin (doublet polypeptides over 500 kD each) [Maher et al., 1985], and Z-nin (~300-400 kD)

[Suzuki et al., 1985]. Z-nin, however, is prepared from myofibrils by use of a proteolytic enzyme [Suzuki et al., 1981], and may be a breakdown product of [Furst et al., 1988].

Several early reports suggested that a third longitudinal filament system, in 11 addition to the thick and thin filaments, exists in striated skeletal muscle cells

[Huxley, 1954; McNeill and Hoyle, 1967]. Superthin (~2-6 nm in diameter) filaments, which supposedly connect the thick filaments to the Z-line and/or span the A-l "gap" junction in overstretched muscle, were described [Sjostrand,

1962; Locker and Leet, 1975]. Wang and associates [Wang et al., 1979;

Wang and Williamson, 1980] subsequently identified, by conducting SDS-PAGE with low percentage acrylamide, three very large proteins in skeletal muscle.

The larger two {doublet proteins) were named titin, and comprise -10% of total myofibrillar protein [Wang et al., 1979]. The titin doublet, called T, and

Tg, have molecular weights of about 2.8 million and 2.4 million, respectively

[Kurzban and Wang, 1988]. The smaller titin polypeptide, Tg, is thought to be a proteolytic product of T, [K. Wang, 1985]. Tj can be purified without use of denaturing solvents, and is sometimes called native titin [Trinick et al., 1984;

Wang et al., 1984]. Titin is a very long (~ 1.2 A/m), thin (-4-5 nm in diameter), extensible molecule [Trinick et al., 1984; Wang et al., 1984; Nave et al., 1989], and extends from the M-line to the Z-line in each half of the sarcomere [Furst et al., 1988, 1989]. The extra part of titin in T,, which is absent in Tj, is located at the Z-line [Furst et al., 1988]. This domain is also the C-terminal domain of intact titin [S.-M. Wang et al., 1991], and this part of titin can be considered as an integral Z-line protein. It is now thought that the

"gap" filaments seen in the region between the ends of thick and thin filaments in overstretched muscle [Locker and Leet, 1975] are part of the titin filaments 12 [LaSalle et al., 1983; Robson and Huiatt, 1983; Maruyama et al., 1989]. Thus,

titin composes a third, elastic filamentous system in the sarcomere. Titin may

serve as a template for thick filament assembly during muscle development and

maintain sarcomeric registry in developed striated muscle cells [Wang et al.,

1979; K. Wang, 1985; Horowits et al., 1986; for a recent review, see Fulton

and Isaacs, 1991]. K. Wang et al. [1991] very recently have provided evidence

that titin filaments play a major role in muscle elasticity and that skeletal muscle

cells may mediate their sarcomeric stiffness/elasticity by selective expression of

specific titin isoforms. Recent cDNA-derived primary structural studies have

found that titin contains two regular motifs, which resemble those also found in

kinase and C-protein [Labeit et al., 1990], two proteins

known to interact with myosin. This adds support for the notion that part of

the titin molecule can interact with myosin. Although "titin" is used most often

as the name for this giant protein [Fulton and Isaacs, 1991], this protein also is

sometimes referred to as "connectin" [Maruyama et al., 1981].

The third band discovered by Wang et al. [1979] by SDS-PAGE (~500 kD), and initially called band 3, was subsequently named [Wang, 1981].

Antibody localization studies originally indicated that nebulin was located at the

Ng line in the l-band [Wang and Williamson, 1980]. More recent studies, however, suggest that nebulin may comprise a fourth filamentous system in skeletal muscle , and that nebulin extends from approximately the free ends of the thin filaments to the Z-lines [Wang and Wright, 1988]. Nave et 13 al. [1990] have shown that nebulin interacts in vitro with o-actinin. The C-

terminal end of nebulin is located at the Z-line [Jin et al., 1990] and, thus, this

part of nebulin also can be considered to be an integral Z-line protein. Jin and

Wang [1991] have demonstrated by co-sedimentation and Western blot

analysis that cloned fragments of nebulin also will bind to F-actin. A single

nebulin molecule is certainly large enough (~800 kD) [Hu et al., 1989] to

extend the full length of the thin filament. It has been proposed [Jin and Wang,

1991; Labeit et a!.,1991] that nebulin might act as a giant ruler or actin-binding

template for thin filaments in skeletal muscle cells. Interestingly, nebulin is

absent in cardiac and smooth muscles [Hu et al., 1986; Wang and Wright,

1988].

Overall Vertebrate Cardiac Muscle and Smooth Muscle Structure

Vertebrate cardiac muscle cells, like skeletal muscle cells, are striated and contain myofibrils arranged in register [Sommer and Johnson, 1979; Forbes and

Sperelakis, 1984]. muscle cells, however, are innervated by the autonomic nervous system. Each cardiac muscle cell is in tight contact with adjacent cells in a fiber, and has specialized cell-cell contact regions called

"intercalated disks". These unique structures serve as electrical coupling sites between cells and, thus, permit the heart to act as an anatomic syncytium.

The actin filaments of the terminal myofibrillar sarcomeres in cardiac muscle cells are attached at the intercalated disk [Yamaguchi et al., 1988]. Thus, this 14 region also functions, in a sense, like a Z-line structure. The specialized

structure of the intercalated disk is tripartite in nature and contains: (1) gap

junctions (responsible for the electrical coupling of heart cells), (2) desmosomes

(IF attachment sites), and (3) fascia adherens (actin attachment sites for the

terminal sarcomeres). Z-line proteins such as a-actinin [Tokuyasu et al.,

1983a], filamin [Koteliansky et al., 1986], desmin [Kartenbeck et al., 1983;

Tokuyasu, 1983; Tokuyasu et al., 1983b; Bilak et al., 1991b], zeugmatin

[Maher et al., 1985], and titin [Furst et al., 1988], as well as

[Mueller and Franke, 1983], also are found at the intercalated disk.

Smooth muscle is innervated by the autonomic nervous system and, like cardiac muscle, is referred to as involuntary muscle. Smooth muscle cells are spindle-shaped, thicker in the middle, and tapered at the ends [Burnstock,

1970]. Each cell has a single centrally-located nucleus. When viewed by polarized light microscopy, no regular banding pattern is evident, i.e., the cells and overall muscle appear smooth, and not striated [for reviews, see Somylo et al., 1984; Bagby, 1986]. When viewed by electron microscopy, the smooth muscle contractile apparatus contains visible, interdigitating thick (myosin- containing) and thin (actin-containing) filaments which are somewhat analogous to those of striated muscle [Kargacin et al., 1989]. In smooth muscle cells, the thick and thin filaments differ in their ratio to each other (~ 11-15 thin filaments surround each thick filament, in contrast to 6 thin filaments in striated muscle)

[Bois, 1973; Ashton et al., 1975]. Thin filaments are longer (up to ~4.5 //m in 15 length) [Small et al., 1990] than those of striated muscle cells. Because the

myosin filaments are side-polar, no central bare zone is observed [Craig and

Megerman, 1977; Cooke et al., 1989]. Smooth muscle cell thick filaments do

not contain C-protein or M-proteins. The thin (actin) filaments contain

tropomyosin, caldesmon, and calponin, but not troponin [for a review, see

Trybus, 1991]. A distinguishing feature of smooth muscle cells is the presence

of cytoplasmic- and membrane-associated dense bodies (CDBs and MADBs,

respectively) [Bagby, 1986; Stromer and Bendayan, 1988]. These structures

are observed as electron-dense areas which are located along the plasma membrane (MADBs) and within the sarcoplasm (CDBs), and are thought to be analogs of the Z-line of striated muscle cells because the thin filaments are anchored to them [Ashton et al., 1975] and because a-actinin (a major Z-line protein of striated muscle) is one of their major protein components

[Schollmeyer et al., 1976]. The dense bodies also contain zeugmatin, as is the case for skeletal and cardiac muscle Z-lines [Maher et al., 1985].

Release of Ca^^ into the cytoplasm leads to smooth muscle contraction

[Small, 1977; Guyton, 1986]. Unlike skeletal muscle, smooth muscle contraction results not just from a nerve impulse, but also in response to hormonal stimulus or stretch. Smooth muscle contraction is slower than striated muscle contraction, and the smooth muscle cell can remain contracted for a longer period of time [Kamm and Stull, 1985a]. Within the smooth muscle cell, contraction proceeds by a sliding filament mechanism that is controlled 16 primarily by a Ca^+- dependent system [Kamm and Stull, 1985b]. A

third filamentous system, the IPs, are abundant in smooth muscle cells [Small

and Sobieszek, 1977; Stromer and Bendayan, 1988]. They somehow are

attached to the dense bodies [Uehara et al., 1971; Ashton et al., 1975;

Stromer and Bendayan, 1988], but the exact arrangement of IPs in smooth

muscle cells remains an active area of research. The major protein subunit of

IPs in smooth muscle cells of the digestive system is desmin [Huiatt et al.,

1980]. In some smooth muscle cells of the vascular system, is the

sole, or major, IP protein, but in other cells, desmin is

the sole, or major, IP protein detected [Osborn et al., 1981, 1987; Pujimoto et

al., 1987]. Proposed functions for IPs in smooth muscle cells include

organization of dense bodies and the contractile apparatus [Uehara et al., 1971;

Ashton et al., 1975] and positioning of the nuclei and mitochondria [Stromer

and Bendayan, 1990].

Properties of Intermediate Filaments

Intermediate filaments (IPs) represent one of the three major cytoskeletal

filamentous systems present in most eukaryotic cells [Lazarides, 1980]. The term "intermediate-sized filaments" was first used by Ishikawa et al. [1968] to describe conspicuous filaments, -10 nm in diameter, in the cytoplasm of cultured striated muscle cells. IPs are now known to be present in nearly every vertebrate cell type [Lazarides, 1980, 1982; Steinert and Roop, 1988]. 17 Biochemical and immunological studies have identified at least five cytoplasmic

classes (, desmin, vimentin, glial, and ) and one nuclear

class () of IPs [for reviews, see Steinert and Roop, 1988; Robson, 1989;

Stewart, 1990]. The cytoplasmic IPs originally were subdivided and classified

based upon their tissue specific expression in mature cells. The major protein

components of these five classes of IPs are the [a complex group of

~30 proteins that can be subdivided into acidic (pi = ~4.5-5.5; -40-60 kD)

and basic (pi = ~ 5.5-7.5; ~ 50-70 kD) subsets], which comprise the keratin IPs

of epithelial cells; desmin (~53 kD), which forms the IPs in differentiated

striated muscle cells, the smooth muscle cells of the digestive and urogenital

tracts, and many, but not all, of the smooth muscle cells of the vascular

system; vimentin (~54 kD), which forms the IPs in fibroblasts and other

mesenchymally-derived cells; glial fibrillarv acidic protein (GPAP) ("-51 kD),

which forms the IPs of the astrocytes and glial cells; and, the neurofilament

triolet polvpeptides f-60-70 kD (NP-L), -105-110 kD (NP-M), and -135-150

kD (NP-H)], which comprise the IPs in neurons [Lazarides, 1980; Geisler and

Weber, 1982; Robson, 1989]. The lamins (including lamins A, B, and C; -60-

70 kD), which comprise the nuclear lamina just inside the inner membrane of the nuclear envelope, were later added as a sixth class of IPs [Gerace, 1986;

Robson, 1989]. Although this tissue-specific classification scheme for IPs is generally useful, many exceptions to it exist because some cells express more than one type of IF protein [Lazarides, 1982]. Desmin and vimentin, for 18 instance, are botli present in some vascular smooth muscle cells [Schmid et al.,

1982; Osborn et al., 1987]. Many cells grown in culture express vimentin in

addition to the characteristic IF protein of the cell. Likewise, cells in early

developmental stages often coexpress vimentin and one or more other IF

proteins [Lazarides, 1982; Gustafsson et al., 1989]. As an example of the

latter case, vimentin and desmin are both expressed in early progenitor cells of

skeletal muscle (myoblasts and myotubes), but, during development, vimentin

expression is turned off and only desmin is present in fully differentiated

skeletal [Bennett et al., 1979; Tokuyasu et al., 1985; Bilak et al.,

1987]. , a recently identified IF protein in cells of the central nervous system [Lendahl et al., 1990; Steinert and Liem, 1990], is initially coexpressed with vimentin in neuro-stem cells, but as neurogenesis progresses, nestin is replaced by o- [Fliegner et al., 1990]. Expression of a-internexin and vimentin both then decrease with continued development and the expression of the type IV neurofilament triplet proteins increases. Only a-internexin and the neurofilament triplet proteins are expressed in terminally differentiated neurons of the central nervous system [Pachter and Liem, 1985; Kaplan et al., 1990].

The sixth major subclass of IFs, the lamins. are especially interesting [for a review, see Dessev, 1990]. The lamins are the major constituents of the fibrous lamina that lies next to the inner membrane of the nuclear envelope

[Gerace and Blobel, 1980]. The fibers (filaments) in the lamina have a diameter of about 10 nm and often show a characteristic tetragonal order [Aebi et al., 19 1986]. It was not until fairly recently [for reviews, see Aebi et al., 1986;

Gerace, 1986; Franke, 1987; Robson, 1989], that it became evident that the

lamins share so many properties in common with cytoplasmic IFs that they

were added to the list of IF family members. The major lamins of mammalian

cells included A, lamin B, and lamin C [Gerace and Blobel, 1980; Steinert

and Roop, 1988], although recent studies [Kaufmann, 1989; Hoger et al.,

1990] suggest there are several additional lamins. All lamins are capable of

forming reconstituted IFs, —10 nm in diameter, in vitro [Aebi et al., 1986]. The

lamins expressed in a wide range of cells and species are homologous [Hoger et

al., 1990]. Lamins A and C are very similar in primary sequence, and are

synthesized from the same , but from two messages obtained by

[McKeon et al., 1986; Fisher et al., 1986]. Lamin B shares

less homology, and is encoded by a different gene [Hoger et al., 1988]. It has

been suggested that the cytoplasmic IF multigene family evolved from a

common ancestral lamin gene [Franke, 1987; Weber et al., 1989]. The

individual roles of the lamins remain unclear, but collectively they are thought to

provide a structural framework for the nuclear envelope and anchoring site for nuclear chromatin in interphase cells [Burke and Gerace, 1986]. Lamin B is more tightly associated to the nuclear membrane than lamins A and C and, thus, appears to play a significant role in attachment of the lamins to the inner nuclear membrane [Gerace and Burke, 1988; Dessev, 1990]. In addition, it has been shown that lamin B interacts in vitro with the C-terminal region of 20 vimentin and desmin [Georgatos and Blobel, 1987b; Georgatos et al., 1987],

and the nucleus may serve as a nucleation site for IF assembly [Albers and

Fuchs, 19891. It has long been suggested that IPs may play a role in

communication between the plasma membrane and the nucleus [for a review,

see Goldman et al., 1986], but details of this process remain to be established.

Based upon all of the sequence data and comparison of

homologies obtained for members of the IF family in the past few years, an

alternative classification scheme for the IF proteins has been developed [for reviews, see Steinert and Roop, 1988; Robson, 1989]. In this scheme, types I and II represent acidic and neutral/basic keratins, respectively. Desmin, vimentin, GFAP, and a more recently discovered IF protein in peripheral neurons, [Leonard et al., 1988; Parysek et al., 1988], are grouped into type III IF proteins. The neurofilament triplet proteins (NF-L, NF-M, and NF-

H) are classified as type IV, and the lamins are classified as type V IF proteins

[Steinert and Roop, 1988; Robson, 1989]. Nestin, a recently described protein in neuro-stem cells, may represent a type VI sequence class [Lendahl et al.,

1990; Steinert and Liem, 1990]. All of the IF proteins share some common structural properties and principles in that they have: (a) an amino-terminal, fairly short, non-alpha-helical head domain of variable length and amino acid sequence, (b) a central, highly alpha-helical rod domain consisting of approximately 310-315 amino acids (~356 for the nuclear lamins), and (c) a non-alpha-helical carboxy-terminal tail domain of variable length and amino acid 21 sequence [Geisler and Weber, 1982; Steinert and Roop, 1988; Pieper et al.,

1989]. The central, alpha-helical rod domain (38 kD) is highly conserved among

the IF subunits. The degree of this homology is greatest within a given class or

sequence type (e.g., desmin and vimentin). It is because of the homologous

rod domain that all (either alone or in certain combinations) IF proteins assemble

into nearly identical-looking filaments in physiological-like solutions. Major

differences observed in the IF subunits (such as molecular mass, pi, and

solubility) are due in large part to the variability in the two end domains, and

especially in the case of molecular mass, to the differences in the carboxy-

terminal tail domain.

IFs arise ultimately by assembly from monomeric protein subunits. Current

models suggest that a basic building block of IFs is a coiled-coil dimer, which is

formed from two IF polypeptides paired through their alpha-helical rod domains

in a side-by-side, non-staggered fashion [Ip et al., 1985a,b; Parry et al., 1985;

Quinlan et al., 1986; Steinert, 1990]. Two of these dimers then align side-by- side to form an ~ 2-2.5 nm wide x ~ 50-60 nm long tetramer (a fairly stable assembly intermediate) [Geisler and Weber, 1982; Pang et al., 1983], called a protofilament [Stromer et al., 1981; Ip et al., 1985a,b], but the exact arrangement of the two dimers in the protofilament remains unclear. Geisler et al. [1985] reported that the dimers are arranged in an anti-parallel fashion, but not all investigators [Ip, 1988; Birkenberger and Ip, 1990] agree with this suggestion. Whether the two dimers are in register [Ip et al., 1985a,b; 22 Hisanaga et al., 1990], or are staggered with a slight offset [Stewart et al.,

1989; Potschka et al., 1990] also remains controversial. The next step in

assembly is thought to be formation of an octamer {~4-4.5 x 70 nm), called a

protofibril (Aebi et al., 1988] or a subfilament [Stromer et al., 1981]. The

precise structure of the octamer is unknown, but it may be composed of two

protofilaments arranged in a half-staggered fashion [Ip et al., 1985a,b]. Further

assembly involves both side-to-side and end-to-end aggregation of assembly intermediates as the ~ 10 nm diameter, long (> 1 //m) IPs are assembled [Ip et al., 1985a,b; Aebi et al., 1988].

While it is generally agreed that the different IF proteins assemble into morphologically similar filaments by virtue of the homologous rod domains, the roles of the end domains are less clear. The amino-terminal head domain apparently plays a significant role in controlling filament assembly/disassembly

[Traub and Vorgias, 1983; Kaufmann et al., 1985]. For instance, proteolytic cleavage [Kaufmann et al, 1985], or deletion by genetic manipulation [Quinlan et al., 1989; Ratts et al., 1990], of the amino-terminus in type III proteins inhibits their ability to assemble into filaments. Phosphorylation by specific protein kinases of the amino-terminal head dolTiains can cause disassembly of

IPs and inhibit assembly of desmiri or vimentin into IPs [Inagaki et al., 1987,

1988; Geisler and Weber, 1988; for reviews, see Robson, 1989; Skalli and

Goldman, 1991]. Likewise, the lamin disassembles as the result of phosphorylation and reappears as a function of dephosphorylation during 23 mitosis [Dessev, 1990]. The precise role of the C-terminal tail domain of IF

proteins in filament assembly is unclear [Quinlan et al., 1989; Hatzfeld and

Weber, 1990].

IPs radiate throughout the cytoplasm of cells and seem to terminate at both

the nuclear and plasma membranes [Goldman et al., 1986; Robson, 1989].

Evidence of specific associations of the two end domains of IF proteins with

protein components located at these two membrane sites is accumulating [for a

review, see Robson, 1989]. The C-terminal ends of both desmin and vimentin

tetramers will interact with lamin B [Georgatos and Blobel, 1987b; Georgatos et

al., 1987]. Specific binding between the amino-terminal ends of desmin or

vimentin with ankyrin (a protein located in the plasma membrane skeleton) also

has been shown [Georgatos and Marches!, 1985; Georgatos and Blobel, 1987a;

Georgatos et al., 1987]. Georgatos and associates [Georgatos and Blobel,

1987a; Georgatos et al., 1987] have hypothesized that the IFs are vectorially

arranged in vivo, having specific binding sites at both the nuclear and plasma

membrane surfaces.

Despite considerable advances regarding the biochemical and structural properties of IF proteins, IFs remain a poorly understood cytoskeletal system

with regard to function [Franke, 1987; Bloemendal and Pieper, 1989;

Klymkowsky et al., 1989]. In the past, IFs were often considered as rather

"static" structures that fulfilled cytoskeletal or mechanical roles, such as maintaining cell shape and positioning of subcellular organelles [for a review. 24 see Skalli and Goldman, 1991]. The discovery that phosphorylation and

dephosphorylation greatly alters IF stability [Inagaki et al., 1987], however, has

led to the general assumption that IFs are much more dynamic components of

cells [Lamb et al., 1989; Tao and Ip, 1991; Skalli and Goldman, 1991]. That a

few cells appear not to have an IF cytoskeleton [Venetianer et al., 1983; Traub

et al., 1983; Hedberg and Chen, 1986] and that many cultured cells seemingly

function fairly normally after IF disruption [Schultheiss et al., 1991], however,

have left both their role and importance as enigmas.

The major IF protein of most differentiated muscle cells is desmin [for reviews, see Robson et al., 1984; Stromer, 1990]. Proliferating and postmitotic myoblasts express vimentin, with desmin absent until the postmitotic myoblast stage [Babai et al., 1990]. In the case of skeletal muscle cells, there is a dramatic increase and decrease in synthesis of desmin and vimentin, respectively, following myoblast fusion [Bennett et al., 1979;

Lazarides et al., 1982]. The exact role of IFs in developing muscle cells remains unclear [Schultheiss et al., 1991; Tao and Ip, 1991]. The amount of desmin present in adult smooth, cardiac, and skeletal muscle cells varies. Desmin comprises about 8% of the myofibrillar protein fraction in adult smooth muscle of the digestive system [Huiatt et al., 1980], but only about 1-2% of the myofibrillar protein fraction in adult cardiac [Hartzer, 1984], and about 0.35% of the myofibrillar protein fraction in adult skeletal muscle [O'Shea et al., 1981].

Desmin has been purified from all three muscle types [Huiatt et al., 1980; 25 O'Shea et al., 1981; Hartzer, 1984]. In general, high salt buffers are used to

first extract the actomyosin, leaving behind a cytoskeletal, IF-enriched residue.

The desmin can be extracted/solubilized from the residue by using solutions low in pH (<4) [O'Shea et al., 1979, 1981] or containing denaturing agents such as

6 M urea [Huiatt et al., 1980; for a review, see Robson et al., 1981].

Purification is then achieved by conventional chromatographic techniques conducted in the presence of urea [Huiatt et al., 1980; O'Shea et al., 1981].

Purified desmin will remain soluble in low ionic strength, slightly alkaline buffers after removal of urea, and will assemble into synthetic IPs by dialysis against buffers near physiological ionic strength and pH [Huiatt et al., 1980; Stromer et al., 1987; Chou et al., 1990].

The major role of desmin IPs in adult skeletal muscle cells is thought to be cytoskeletal in nature, i.e., to maintain lateral registry of the myofibrils by providing a framework which links adjacent myofibrils together [Lazarides,

1980; Richardson et al., 1981] and the outer layer of myofibrils at their Z-line levels to the sarcolemma [Bilak et al., 1991a]. Antibody localization studies at the light microscope level indicate that desmin is located primarily at the periphery of the myofibrillar Z-lines forming a "honeycomb-like" network when viewed in cross-section [Granger and Lazarides, 1978]. Reports of unambiguous identification of IPs in adult striated muscle by electron microscopy, however, have been rare [see discussions in Bennett et al., 1979;

Richardson et al., 1981]. And, most identification and localization studies of 26 IFs in adult striated muscle have been done with avian muscle. Richardson et

al. [1981] conducted the first electron microscope, immunolocalization studies

of desmin IFs involving adult striated muscle. Their desmin antibodies labeled

filamentous structures in the intermyofibrillar regions at the Z-line level of

chicken cardiac and skeletal muscle myofibrils (i.e., a transversely-oriented

pattern). These results were subsequently confirmed in avian muscle by

Tokuyasu and associates [Tokuyasu et al., 1983a,b]. Tokuyasu [1983], using

frozen sections of chicken cardiac muscle, also identified some longitudinally-

oriented IFs in the intermyofibrillar spaces, in addition to the transversely-

oriented IFs located at the Z-line levels. IFs have also been identified that

connect the myofibrillar Z-lines to mitochondria [Tokuyasu et al., 1983b; Bilak

et al., 1991a], nuclei [Ferrans and Roberts, 1973; Forbes and Sperelakis, 1980;

Tokuyasu et al., 1983a; Bilak et al., 1991a], and the sarcolemma [Ferrans and

Roberts, 1973; Pierobon-Bormioli, 1981; Bilak et al., 1991a] in adult striated muscle cells.

On the basis of the above identification and localization evidence, IFs in mature striated muscle cells appear to somehow attach to the myofibrillar Z- lines. However, direct structural [Tokuyasu et al., 1985; Bard and Franzini-

Armstrong, 1991; Bilak et al., 1991a,b] or biochemical evidence demonstrating direct, or even indirect, attachment of the IF network to the myofibrillar Z-lines has been difficult to obtain. Bilak et al. [1991a] recently have shown that the

IFs come close enough (~8 nm) to the outer edge (periphery) of the myofibrillar 27 Z-lines that either the C-terminal end domains of desmin, or perhaps an

additional IF/myofibril crosslinking protein, should be able to easily span this

distance. Tokuyasu et al. [1985] actually suggested that this crosslinking role

may be mediated by one or more yet-to-be identified crosslinking protein(s).

Isobe et al. [1988] have suggested that a-actinin may be involved in association

of IPs and actin filaments in cultured hamster cardiac cells, but the evidence is

unconvincing. More evidence, involving both biochemical and structural

studies, is needed. Possible protein candidates that may serve as a link

between IPs and myofibrils will be discussed in the next section.

Intermediate Filament-Associated Proteins (IPAPs)

IPAPs were first recognized as proteins which copurify with and/or colocalize

with IF proteins, and were studied primarily because of their potential roles in

organization of IPs and linkage of IPs to other cellular components. In comparison to the IP proteins, most of these proteins have not been studied in much detail. Approximately 15 to 20 putative IPAPs have been identified [for reviews, see Steinert and Roop, 1988; Robson, 1989; Poisner and Wiche,

1991]. Steinert and Roop [1988] have assigned the IPAPs to four classes based upon their size and possible function; (a) small (~ 10-45 kD) IPAPs that bind IPs laterally into macrofilament complexes (e.g., filaggrins), (b) large

(> ~200 kD) IPAPs that cross-link IPs into loose networks (e.g., synemin, ), (c) IPAPs that seemingly function as capping proteins (e.g., ankyrin. 28 lamin B, ), and (d) other IFAPs that don't seem to fit into the first

three classes (e.g., epinemin, yff-internexin).

One set of rather well characterized IFAPs are the filaggrins, ~ 26-49 kD

histidine-rich basic proteins that are specifically expressed in epidermal cells.

First isolated from the stratum corneum of mammalian epidermis [Dale, 1977;

Steinert et al., 1981], the filaggrins are involved in cross-bridging keratin IFs

into macrofibrils [Steinert et al., 1981]. The filaggrins differ considerably in size

depending upon species of origin (e.g., mouse = ~26 kD, human = ~35 kD)

[for a review, see Traub, 1985]. The filaggrins also interact in vitro with

desmin and vimentin, possibly through their rod domains, causing formation of

filaments into the macrofibers [Dale et al., 1978; Steinert et al., 1981]. The physiological significance of this latter result, however, is unknown since filaggrins are unique to epithelial tissue. Interestingly, the filaggrins are first synthesized as a large molecular weight (~400-500 kD), highly phosphorylated precursor molecule called profilaggrin [Meek et al., 1983; Rothnagei and

Steinert, 1990]. The profilaggrin consists of many tandemly arranged repeats, which are subsequently cleaved post-translationally into many copies of the small filaggrins [Rothnegel and Steinert, 1990].

Another fairly well characterized IFAP is called plectin [Wiche et al., 1982; for a review, see Wiche, 1989] or IFAP-300 [Yang et ai., 1985]. Plectin originally was identified as an ~300 kD polypeptide present in the vimentin- enriched cytoskeletal residue prepared from rat glioma C6 or human HeLa 29 cultured cells [Wiche et al., 1982]. Plectin subsequently was isolated from

other sources such as BHK-21 (fibroblast) cell lines [Lieska et al., 1985] and

from bovine lens tissue [Weitzer and Wiche, 1987], and characterized

[Herrmann and Wiche, 1987; Foisner and Wiche, 1987; Weitzer and Wiche,

1987; Wiche et al., 1991]. By using indirect immunofluorescence and

immunoelectron microscopy studies, plectin has been found in ~ 20 mammalian

cell lines and various mammalian cell types and tissues, including smooth,

cardiac, and skeletal muscle [Wiche et al., 1983]. In smooth muscle cells,

plectin is concentrated at the cell periphery. In cardiac muscle cells, plectin is

located at the intercalated disks and, as in skeletal muscle cells, Z-lines [Wiche

et al., 1983]. Immunogold electron microscopy and quick-frozen, deep-etched

electron microscopy have shown that, in fibroblasts, plectin is associated with

vimentin filaments at their intersection or cross-over points [Foisner et al.,

1988a]. In vitro studies, in which vimentin filaments were assembled in the

presence of plectin and rotary shadowed, confirmed these observations [Foisner

et al., 1988a; Foisner and Wiche, 1987].

Solid-phase binding assays have demonstrated that plectin binds via its own rod domain to the rod domain of all type III and type IV IF proteins. In addition, plectin binds to the -associated proteins, MAP-1 and MAP-2, and spectrin-like (spectrin, fodrin) polypeptides in solid-phase binding assays

[Herrmann and Wiche, 1987; Foisner et al., 1988a,b, 1989]. Moreover, plectin binds in vitro to lamin B (Foisner et al., 1991]. Plectin's ability to interact with 30 IPs is regulated by phosphorylation, having domains specific for cAMP-

independent, cAMP-dependant, and Ca^+/calmodulin-dependent kinases

[Herrmann and Wiche, 1983, 1987]. Because plectin is widespread and has

the abilities to crosslink IPs, to interact with and ,

and to interact with other components that may interconnect IPs to the plasma

and nuclear membranes, plectin has been considered as a universal cytoplasmic

crosslinker [Herrmann and Wiche, 1987; Poisner et al., 1989, 1991; Wiche,

1989; Poisner and Wiche, 1991].

Electron microscopy (shadowing and negative staining) combined with

biochemical and biophysical analyses reveal that native plectin is a dumbbell

shaped tetramer (total mass of 1,200 kD by SDS-PAGE) with two structurally

and functionally distinct domains [Weitzer and Wiche, 1987; Poisner and

Wiche, 1987]. Two globular head domains (~9 nm in diameter) are joined by a

long (2 nm in diameter by -190 nm in length) rod domain consisting of a

double-stranded, a-helical coiled-coil [Poisner and Wiche, 1987; Wiche et al.,

1991]. Plectin molecules self-associate, either intra- or inter-molecularly, via

the globular end domains [Poisner and Wiche, 1987]. That plectin is a rod shaped molecule, flanked by globular domains, has recently been supported by cDNA derived primary sequence data [Wiche et al., 1991]. The size calculated from the sequence (~466 kD) is larger than calculated for plectin by SDS-PAGE

(300 kD). Whether this difference in size is due to anomalous electrophoretic mobility, or to degradation of plectin is not yet known [Wiche et al., 1991]. 31 Synemin was originally identified by SDS-PAGE as a 230 kD polypeptide

present in IF-enriched fractions from chicken gizzard smooth muscle [Granger

and Lazarides, 1980]. Synemin's presence subsequently was detected, by

using immunoautoradiography, in embryonic and adult avian smooth, skeletal,

and cardiac muscle [Sandoval et al., 1983]. Price and Lazarides [1983], however, subsequently reported in the same year that synemin was absent, as detected by antibody localization, from adult chicken cardiac muscle. The discrepancy between these two findings, both from the same laboratory, was not discussed in the reports, but may have resulted from the two different techniques utilized. Indirect immunofluorescence studies, using rabbit polyclonal antibodies raised against avian gizzard synemin, showed that synemin's distribution in cultured skeletal muscle cells was indistinguishable from that of desmin and vimentin during , being present throughout the cytoplasm in early myogenesis, and coalescing with desmin IPs at the myofibrillar Z-line periphery as the cells matured [Granger and Lazarides, 1980;

Lazarides et al., 1982]. In mature skeletal muscle cells, synemin labeling was localized at the periphery of myofibrillar Z-lines [Granger and Lazarides, 1980].

Synemin subsequently was found associated with vimentin IPs from both avian erythrocytes [Granger and Lazarides, 1982] and chicken eye lens cells

[Granger and Lazarides, 1984]. Double-immunofluorescence labeling studies demonstrated that the synemin colocalizes with the vimentin-containing IPs in these two cell types. Electron microscopy of low-angle shadowed specimens 32 revealed that anti-synemin antibodies bound in discrete, periodically spaced foci along the length of the vimentin filaments. The periodicity of the synemin antibodies decreased from an average of 230 nm in embryonic erythrocytes to

180 nm in mature erythrocytes, suggesting that possible conformational changes within the IPs occur during development (erythropoiesis) [Granger and

Lazarides, 1982]. Anti-synemin labeling also was quite noticeable where two vimentin IPs came into close lateral proximity or where two IPs intersected, suggesting that synemin may crosslinit adjacent IPs [Granger and Lazarides,

1982, 1984; Lazarides and Granger, 1983]. Comparative studies, albeit limited

In scope, suggested that synemin from avian erythrocytes and from avian smooth and skeletal muscle are highly homologous if not identical [Granger et al., 1982]. Thus, synemin is associated with desmin- and vimentin-containing

IPs from both muscle and non-muscle cells. However, synemin does not occur in all cell types containing these IP proteins; for example, synemin is not present in fibroblasts or proliferating myoblasts [Granger and Lazarides, 1980; Lazarides et al., 1982].

The amount of synemin, as determined by densitometric analysis of SDS- polyacrylamide gels of IP-enriched fractions, is approximately one-fiftieth to one-hundredth (mole/mole ratio) the amount of desmin present in avian smooth muscle [Granger and Lazarides, 1980] or of vimentin present in avian erythrocytes [Granger et al., 1982]. Synemin has been purified from chicken gizzard smooth muscle and partially characterized [Sandoval et al., 1983]. 33 Synemin has a relatively high content of acidic (especially glutamic) amino acid

residues (pi of synemin = -5.4) and of serine residues. Synemin, in cell

fractions [Granger and Lazarides, 1980] and during and after purification

[Sandoval et al., 1983], is highly susceptible to proteolysis. Synemin is

phosphorylated in smooth muscle and, thus, is a phosphoprotein. The high

content of serine may account for synemin's ability to incorporate exogenously added organic phosphate [Sandoval et al., 1983]. These workers also reported that the soluble form of synemin, in physiological !:ke solutions, is a globular tetramer (~980 kD) [Sandoval et al., 1983]. They also reported that synemin binds to reconstituted desmin filaments and to soluble desmin. This latter conclusion resulted from experiments in which immunoprecipitation of desmin by anti-desmin antibodies was inhibited when pre-incubated with synemin.

They also reported that synemin has no effect on the rate or extent of desmin filament assembly [Sandoval et al., 1983]. Moon and Lazarides [1983] suggested, based upon pulse labeling studies in avian erythroid cells, that vimentin assembles into IPs first, before synemin can be incorporated into these filaments; thus, the rate of synemin assembly was dependent on vimentin assembly. The role of synemin is unknown, but it has been suggested that synemin may regulate the extent of crosslinking of IPs into loose networks and/or attach IPs to other intracellular organelles or the plasma membrane

[Granger and Lazarides, 1984]. Other than the studies that will be described in this dissertation, all studies reported to date on synemin have been conducted 34 in the laboratory of Dr. Elias Lazarides at the California Institute of Technology.

The same laboratory that discovered synemin also identified a protein with a

molecular weight of ~ 280,000 in embryonic skeletal muscle, and named it

paranemin [Breckler and Lazarides, 1982]. Paranemin was codistributed with

desmin, vimentin, and synemin in all developing avian myogenic cells that they

examined. The expression of paranemin seems to be developmentally

regulated, as evidenced by a continuous decrease in expression during

development to non-detectable levels in adult avian smooth and skeletal

muscles [Breckler and Lazarides, 1982; Price and Lazarides, 1983]. However,

paranemin is present in adult avian cardiac muscle, where it supposedly

replaces synemin that reportedly is not expressed [Price and Lazarides, 1983].

As demonstrated by double immunofluorescence microscopy, paranemin has the same cytoplasmic distribution as desmin, vimentin and synemin (when they are present) in myogenic cells (e.g., post-mitotic myoblasts and both early and late myotubes) and in some avian fibroblasts [Breckler and Lazarides, 1982]. A regulatory, rather than structural, role has been proposed for this protein because its expression is independent of synthesis of other IF proteins and its amount decreases during development in some cells [Breckler and Lazarides,

1982]. Paranemin is a very minor protein in terms of cellular amount [Breckler and Lazarides, 1982] and has not yet been purified and characterized biochemically.

Several other IFAPs that may be involved in organization or function of IPs 35 also are known. Ankyrin and lamin B were described earlier. Little is known

about several others. These include epinemin, an ~45 kD protein associated

with vimentin IPs in various cell types such as epidermis and smooth muscle but

which supposedly does not crosslink them [Lawson, 1983, 1984]; p50, an

~50 kD protein found in BHK-21, PTK1, HeLa, and human astrocytoma cells

[Wang et al., 1983] {epinemin and p50 are structurally and functionally related,

and may be the same protein [E. Wang, 1985]}; a 48 kD protein found in astrocytes [Abd-EI-Basset et al., 1988]; an avian-specific 73 kD protein (NAPA-

73), associated with in mature neuronal cells [Ciment et al.,

1986]; yff-internexin {~70 kD) [Napolitano et al., 1985], identified in a variety of mammalian cells and may be identical to the 70 kD heat-shock cognate and clathrin uncoating ATPase [Green and Liem, 1989]; and, p230, an -230 kD protein associated with plasma membrane from bovine lens cells

[Lehto and Virtanen, 1983]. Microtubule-associated proteins (MAPs 1 and 2) are also sometimes classified as IFAPs because of their ability to cross-link microtubules to neuronal IPs [Leterrier et al., 1982].

Two other poorly characterized IPAPs, which hold considerable interest because of their size and location, are IFAPa-400 and gyronemin. IFAPa-400 is an ~400 kD protein transiently expressed during muscle and neural cell differentiation [Chabot and Vincent, 1990]. Its transient expression during myogenesis, both in vivo and in vitro, parallels the expression of vimentin.

Although present in embryonic skeletal, cardiac, and smooth muscle, and 36 myogenic cells in culture, IFAPa-400 is lost entirely in all but adult vascular

smooth muscle cells [Cossette and Vincent, 1991]. Antibody localization

studies of this avian specific IFAP indicate it is located at IF intersection points;

thus, IFAPa-400 is now classified as a crosslinking IFAP [Cossette and Vincent,

1991]. Of the other IFAPs identified to date, IFAPa-400 seems most similar to

paranemin in pattern of expression, approximate size, and location. Further

work will be necessary to clearly show the degree of similarity of IFAPa-400

and paranemin because it has not been conclusively documented that they are

different proteins. Cossette and Vincent [1991] have proposed that IFAPa-400

is involved in the replacement of IF isoforms (e.g., desmin for vimentin in

myogenic cells) during development.

Gyronemin was identified by using a novel monoclonal antibody that

recognizes the high molecular weight (-380 kD) microtubule-associated protein, MAP-1A, in mammalian brain [Brown and Binder, 1990]. In HeLa cells, this antibody recognizes a unique 240 kD protein (gyronemin) by immunoblot analysis. Labeling with this antibody has also been observed along IFs in a periodic fashion (-436 nm) in a variety of mammalian cells [Brown and Binder,

1990]. Although similar in size to synemin, it seems quite unlikely that gyronemin and synemin are related because gyronemin has a basic pi of -7.7.

The protein a-internexin ( - 66 kD) once was considered an IFAP [Steinert and Roop, 1988], however, primary sequence predictions based on cDNA sequences have now revealed that a-internexin is an actual IF protein (type IV) 37 because it contains the ~310 amino acid residue rod domain of IF proteins

[Fliegner et al., 1990]. The two largest polypeptides of the neurofilaments

triplet (NF-M and NF-H) also were sometimes considered as IFAP candidates

[Steinert and Roop, 1988], but both are now considered as bona fide IF

proteins because primary sequence studies [Geisler et al., 1983, 1984;

Napolitano et al., 1987; Lees et al., 1988] have shown that they also contain

the conserved rod domain of IF proteins. NF-L and NF-M alone as well as

together are capable of forming 10-nm filaments [Balin and Lee, 1991]. NF-H

alone does not efficiently self-assemble into 10-nm filaments [Gardner et al.,

1984; Tokutake et al., 1984; Balin and Lee, 1991], but does so in combinations

with NF-L and NF-M [Balin et al., 1991]. The long, highly phosphorylated carboxy-terminal tail domains of NF-M and NF-H are thought to be responsible

for the lateral projections which appear to cross-bridge NFs [Mulligan et al.,

1991]. Reassembly studies performed on NF subunits indicate that the efficiency, either alone or in combinations, to assemble increases with decreasing size, i.e. NF-H assembles less efficiently vs NF-M which assembles less efficiently vs NF-L [Balin and Lee, 1991]. Antibodies specific for epitopes in the rod domain of NF-L, NF-M, and NF-H failed to bind heteropoiymeric filaments but did recognize the rod domains in all three homopolymers, suggesting a difference in packing of the rod in polymers formed from single NF subunits vs those formed from more than one subunit [Mulligan et al., 1991;

Balin et al., 1991; Balin and Lee, 1991]. Antibodies label the amino- and 38 carboxy-terminal domains of filaments polymerized from any combination of

subunits [Balin et al., 1991].

Explanation of Dissertation Format

This dissertation is arranged using the alternate format. The main body

consists of two full length manuscripts that will be submitted for publication to

different journals. The length and amount of detail given in some sections of

the two manuscripts are longer than will be present when they are submitted

for publication. This has been done to permit a more complete description and documentation of the dissertation research. Each manuscript is formatted according to specific journal instructions and, thus, they differ slightly in style from each other. The work in Section I was done primarily by myself with the exception of confocal and immunofluorescence microscopy, which also involved the technical assistance of Marlene Bright. The experiments in Section II were performed solely by myself, with the advice of Drs. Richard M. Robson and

Marvin H. Stromer. Preceding the papers is a General Introduction, which includes a literature review. The references cited in the General Introduction and the Overall Summary are in the Literature Cited section at the end of the dissertation. 39

SECTION I. SELECTED PROPERTIES OF MUSCLE SYNEMIN AND IDENTIFICATION OF A MAMMALIAN HOMOLOG 40

SELECTED PROPERTIES OF MUSCLE SYNEMIN AND

IDENTIFICATION OF A MAMMALIAN HOMOLOG

Stephan R. Bilak, Richard M. Robson, Marvin H. Stromer, and Ted W. Huiatt

Muscle Biology Group, Departments of Animal Science

and of Biochemistry and Biophysics

Iowa State University

Ames, Iowa 50011, U.S.A

Contact Individual: Dr. Richard M. Robson

Muscle Biology Group, Iowa State University

Ames, Iowa 50011

Telephone: (515) 294-5036

FAX; (515) 294-8181

Running Title: Properties of Synemin and Identification of Mammalian Homolog 41 SUMMARY

We have examined selected properties of synemin (230 kDa) purified from

turkey gizzard smooth muscle by modification of the procedures of Sandoval et

al. (1983), and have identified a synemin homolog in mammalian muscle.

Purified soluble synemin in 10 mM Tris-HCI, pH 8.5, appears spherical (~11 nm

in diameter) as shown by negative stain and low angle shadow electron

microscopy. Chemical crosslinking experiments indicate that soluble synemin contains two 230-kDa subunits. The pH- and ionic strength-dependent solubility properties of synemin are fairly similar to those of desmin, but under physiological-like conditions in which desmin self-assembles into ~10 nm diameter intermediate filaments, synemin self-associates into complex, ~ 15-25 nm diameter globular structures. Results from Western blot analysis, with monospecific polyclonal antibodies against avian synemin, show the presence of the reactive 230 kDa synemin band in adult avian skeletal, cardiac, and smooth muscle samples, and of two reactive bands at ~225 kDa and ~ 195 kDa in adult porcine skeletal, cardiac, and smooth muscle samples. Partial purification of synemin from porcine stomach smooth muscle resulted in fractions highly enriched in the ~225 kDa and ~195 kDa polypeptides as shown by SDS-PAGE and by Western blot analysis. Conventional immunofluorescence and confocal laser scanning microscopy of isolated myofibrils and of frozen sections demonstrated that the synemin homolog is present inside all three adult mammalian muscle cell types and that, in striated 42 muscle cells, the synemin homolog Is colocallzed with desmln at the myofibrillar

Z-llne.

Key words: Intermediate filaments (IPs), -associated proteins (IFAPs), cytoskeleton, myofibrils, synemin 43 INTRODUCTION

Intermediate filaments (IPs), which are about 10 nm in diameter (Ishikawa et

a/., 1968), constitute one of the cytoskeletal filament systems in most

vertebrate cells (Lazarides, 1980). The major protein subunits that comprise

the IPs are from a multigene family, and based on amino acid sequence data

and their degree of homology, the IP proteins can be divided into at least five

major sequence classes (types l-V) (Steinert and Roop, 1988; Robson, 1989;

Stewart, 1990). All IP polypeptides possess a central, a-helical rod domain of

~310 amino acid residues that exhibits considerable homology among the IP

proteins, flanked by non-a-helical terminal domains which vary in length and

amino acid sequence and exhibit little homology (Geisler and Weber, 1982;

Geisler at a!., 1983; Steinert and Roop, 1988).

Desmin, a type III IP protein, is the major component of the IPs in adult striated, visceral and many vascular smooth muscle cells (Schollmeyer at a!.,

1976; Lazarides and Hubbard, 1976; Small and Sobieszek, 1977; Huiatt at a!.,

1980; Stromer, 1990). Although a variety of roles have been proposed for IPs, remarkably little is known with certainty about their function (Klymkowsky at a/., 1989; Skalll and Goldman, 1991). Based primarily upon desmin localization at or near the Z-lines of adult striated muscle cells by immunofluorescence

(Lazarides and Granger, 1978; Granger and Lazarides, 1979) and immunoelectron (Richardson ef a/., 1981; Tokuyasu ataL, 1983a,b; M.M. Bilak era/., 1991a) microscope studies, it has been suggested that desmin IPs form a 44 primarily transversely oriented network at the periphery of the myofibrillar Z-

lines and that the desmin IPs may be involved in maintaining lateral registry of

myofibrils by linking adjacent myofibrils at their Z-lines (Lazarides, 1982). The

nature of the interaction(s) between IPs and myofibrils is not clear. Some

investigators (Tokuyasu et a!., 1985; Bard and Pranzini-Armstrong, 1991) have

suggested that IPs may not even be directly attached to the edge of the Z-lines,

although recent studies (M.M. Bilak, 1991b) suggest that the distance, if any,

between IPs and myofibrillar Z-lines is small enough to easily be spanned by a

crosslinking protein. This raises the question as to whether there are additional

crosslinking components involved in association of IPs and myofibrils.

In addition to the major protein subunits making up the backbone or core of

IPs, many polypeptides have been identified from different cell types that appear to be associated with IPs and that have been named IP-associated proteins (IPAPs) (Steinert and Roop, 1988; Poisner and Wiche, 1991). In comparison to the IP proteins, most of the IPAPs have not been well characterized. Synemin, a 230 kDa polypeptide originally identified in avian smooth muscle because it copurified with desmin during initial preparative steps

(Granger and Lazarides, 1980), has been detected in embryonic and adult avian muscle cells (Granger and Lazarides, 1980; Price and Lazarides, 1983), avian erythrocytes (Granger and Lazarides, 1982; Granger ef a/., 1982), and avian lens cells (Granger and Lazarides, 1984). Indirect immunofluorescence studies showed that synemin's distribution in cultured avian skeletal muscle cells is 45 indistinguishable from that of desmin and vimentin during myogenesis and that

synemin colocalizes with desmin IPs at the myofibrillar Z-line periphery in

mature avian skeletal muscle cells (Granger and Lazarides, 1980; Lazarides et

a/., 1982). That synemin is fairly specific in its distribution (i.e., so far it has

only been found in conjunction with two, type III IF proteins, desmin and

vimentin), and that it has been colocalized with desmin at mature avian skeletal

muscle Z-lines, raises the intriguing possibility that in muscle cells synemin may

crosslink IPs and myofibrils. For synemin to serve as a putative crosslinking

component, it, or a homolog, seemingly should be present in other species.

Synemin, however, is reportedly not present in adult avian cardiac muscle cells,

nor in any mammalian muscle cells (Granger and Lazarides, 1980, 1984; Price,

1987), In this study, we report a new chromatographic protocol for routine

purification of synemin, describe selected properties of the purified protein,

including its molecular appearance and solubility, and present evidence for

synemin's presence and location in adult avian and mammalian muscle cells\

'some of the data presented herein have been presented at the 1990 and 1991 American Society for Cell Biology meetings (S.R. Bilak et al., 1990; M.M. Bilak et al., 1991c) 46 MATERIALS AND METHODS

Purification of Avian Gizzard Synemin-AW steps were carried out at 0-4 °C in

a cold room or on ice unless stated otherwise. The pH adjustment of buffers

was done at the temperature at which the buffer was used. All urea solutions

used in the preparation were made from a stocic 8 M urea solution that had just

been passed through a column of Amberlite MB-3 mixed bed ion exchange resin

(Mallinckrodt Chemical Works, St. Louis, MO) to remove cyanate and heavy metal ion contamination. A 500 g sample of trimmed, fresh turkey gizzard smooth muscle (kept on ice and used within 1 h postmortem) was used to first prepare an acetone powder essentially as described by Sandoval et at. (1983), except that streptomycin (0.01 %, w/v), penicillin (0.01 % w/v), and ovomucoid

(0.01%, w/v) routinely were added to their (1) "buffer A" [140 mM KCI, 2 mM ethylene glycol-bis (yff-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA),

0.1% yff-mercaptoethanol (MCE), 0.2 mM phenylmethyl sulfonyl fluoride

(PMSF), 10 mM Tris-HCI, pH 7.5], (2) "low salt buffer" (10 mM EGTA, 0.1%

MCE, 0.2 mM PMSF, 10 mM Tris-HCI, pH 7.5), and (3) "high salt buffer" (0.6

M Kl, 10 mM NajSjOg, 0.1% MCE, 0.2 mM PMSF, 10 mM Tris-HCI, pH 7.5).

A total of 12 g acetone powder was obtained, and homogenized in 15 volumes

(w/v) of "buffer B" [6 M urea, 2 mM dithiothreitol (DTT), 5 mM Na-p-tosyl-L- arginine methyl ester (TAME), 0.2 mM PMSF, 10 mM Tris-HCI, pH 7.5]

(Sandoval et a!., 1983) by using a 15 s burst at medium speed in a Lourdes homogenizer (Lourdes Instrument Corp., NY). The suspension was stirred for 47

12 h, and then centrifuged at 14,000 x g for 30 min. The supernatant was

saved and the sediment was resuspended in 10 volumes (w/v) of buffer B by

using a 10 s burst at medium speed in the Lourdes homogenizer. After stirring

for 8 h, the suspension was centrifuged at 14,000 x g for 30 min, and the

supernatant was combined with the previous one.

Step I: Hydroxyapatite Chromatography--The combined synemin-containing

urea extract (buffer B) was clarified at 183,000 x p for 1 h and the supernatant

was loaded onto a column of hydroxyapatite (HA-Ultrogel, IBF Biotechnics,

France) that had been poured and equilibrated in buffer B. This particular high

porosity, sturdy resin helped circumvent problems we initially had with low flow

rates with the crude urea extracts. The column was eluted with the following

solutions: (1) 200 ml of buffer B, (2) linear phosphate gradient made with 200

ml of 6 M urea, 2 mM DTT, 5 mM TAME, 10 mM potassium-phosphate, pH

7.5, and 200 ml of the same buffer, but containing 35 mM potassium-

phosphate, (3) 200 ml of 35 mM potassium-phosphate buffer, 6 M urea, 2 mM

DTT, 5 mM TAME, pH 7.5, and (4) a linear phosphate gradient made with 200 ml of 35 mM potassium-phosphate, 6 M urea, 2 mM DTT, 5 mM TAME, pH

7.5, and 200 ml of the same buffer, but containing 200 mM potassium- phosphate. Phosphate concentrations were measured by the colorimetric method of Taussky and Shorr (1953). Eluted fractions were analyzed for the presence of synemin by SDS-PAGE. The pooled synemin-containing fraction was dialyzed overnight against buffer B, (but with 1 mM TAME), applied to a 48 second hydroxyapatite column (Bio-Rad Laboratories, Richmond, CA) (poured

and equilibrated in this buffer), eluted, and analyzed as described above.

Step II: DEAE Sephacel Chromatography-The synenriin-containing fractions

from the second hydroxyapatite column were pooled, dialyzed against "buffer I"

(6 M urea, 2 mM EGTA, 2 mM DTT, 1 mM TAME, 20 mM Tris-HCl, pH 7.5)

and loaded onto a column of DEAE-Sephacel (Sigma Chemical Co., St Louis,

MO) that had been poured and equilibrated in buffer I. The column was eluted

with a linear gradient made from 125 ml each of buffer I and buffer I that

contained 200 mM NaCI. The salt gradient was monitored by titrating aliquots of selected fractions with AgNOg (Robson et ai, 1970). Eluted fractions were analyzed by SDS-PAGE and the synemin-containing fractions were pooled. The

"purified" synemin, normally at a concentration of 0.1-0.3 mg/ml, was concentrated to 1-2 mg/ml by using polyethylene glycol (Sigma) and then dialyzed against 6 M urea, 0.1% (v/v) MCE, 10 mM Tris-HCl, pH 7.5, and stored at -70 °C.

Partial Purification of Porcine Stomach Synem/n-Acetone powder also was prepared from 350 g of fresh porcine stomach smooth muscle as described for the gizzard powder preparation except that the solutions used in the wash steps also contained 1 //M pepstatin A (Sigma). A total of 2 g powder was obtained, and then extracted with buffer B as described for the avian synemin. A total of

180 mg of protein in 190 ml buffer B was loaded onto a column of hydroxyapatite (Bio-Rad) (1.6 x 40 cm) that had been poured and equilibrated in 49 buffer B. Elution was effected with the same order of buffers (volumes of each

were decreased in proportion to the smaller column bed volume) described for

the hydroxyapatite chromatography of the gizzard preparation. Fractions eluting

between 111-144 mM phosphate were pooled and dialyzed against buffer I. A

total of 12.5 mg in 30 ml buffer I was then loaded onto a 1.6 x 40 cm DEAE-

Sephacel column and eluted with a 0-200 mM NaCI linear gradient in buffer I.

Fractions eluting between 155-165 mM NaCI were pooled, dialyzed against 6 M

urea, 0.1% MCE, 10 mM Tris-HCI, pH 7.5, and stored at -70 °C.

Electron Mcroscopz-Samples of purified synemin were thawed and dialyzed

extensively against 10 mM Tris-HCI, 0.1% (v/v) MCE, pH 8.5, and then against

10 mM Tris-HCI, pH 8.5. After clarification at 183,000 x ^ for 1 h, samples

were diluted to 0.1 mg/ml in 10 mM Tris-HCI, pH 8.5, and an aliquot saved for

examination in the electron microscope. Other aliquots of the synemin were

then further dialyzed against 4 changes of either "IF-essembly buffer" (100 mM

NaCI, 1 mM MgClg, 10 mM imidazole-HCI, pH 7.0) overnight, or against 4

changes of 4 M guanidine-isothiocyanate, 0.2 M NaCI, 1 mM DTT, 0.5 mM

EGTA, 100 mM (2-[N-Morpholino] ethanesulfonic acid) (MES) (Sigma), pH 6.5,

for 8 h at 37 °C, and then against 4 changes of the same solution without guanidine-isothiocyanate for 8 h at 37 °C. All samples were examined by negative staining as follows: (1) a drop of the protein solution was placed on to a glow-discharged, carbon-coated 400-mesh grid for 1 min, (2) the grid was gently washed with 30 drops of distilled water, with excess water removed by 50 touching the grid with the edge of a filter paper, (3) the grid was stained with

2% aqueous uranyl acetate for 1 min, excess uranyl acetate was removed with

filter paper, and the grid was air-dried. Some grids of synemin (from 10 mM

Tris-HCI, pH 8.5 samples) obtained from step 2 above were placed into a

vacuum evaporator and unidirectionally shadowed at 8° with 11 mm of Pd/Pt

wire. All samples were examined in a JEOL JEM-100CXII transmission electron

microscope ITEM) operated at 80 kV, and the results were recorded on Kodak

SO-163 electron image film.

Chemical Crosslinking--Pur\f\e6 synemin was dialyzed against 10 mM

triethanolamine-HCI, 0.1% (v/v) MCE, pH 8.5, and then the same solution

without the MCE. After clarification at 183,000 x p for 1 h, the synemin was

crosslinked with glutaraldehyde in either 10 mM triethanolamine-HCI, pH 8.5, or in 65 mM KCI, 0.1 mM DTT, 10 mM triethanolamine-HCI, pH 8.5, at 22 °C.

Glutaraldehyde (25% stock; Aldrich Chemical Co., Milwaukee, Wl), was neutralized to pH 6.8 by addition of barium carbonate just before crosslinking.

Glutaraldehyde was used at a level of either 0.02% (w/v) per 0.05 mg synemin/ml, or twice this level (described in the results). The crosslinking reaction was stopped by adding an equal volume of 10% SDS, 40 mM DTT, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1 M triethanolamine-HCI, pH 8.0

(Hu et a!., 1988), immediately mixing the sample with tracking dye (1:1; v/v)

(Huiatt et aL, 1980), and heating at 100 °C in a Lab-Line Muti-BloK (Melrose

Park, III) for 10 min. Crosslinked and control samples were analyzed according 51 to Weber and Osborn (1969) on 5% acrylamide tube gels (acrylamde:bis-

acrylamide= 50:1, w/w) or on 2% (acrylamideibis-acrylamide = 20:1), 3%

agarose tube gels. Molecular weights were determined from plots of log

versus relative mobility (Weber and Osborn, 1969) obtained by electrophoresis

of crosslinked phosphorylase b (Sigma) molecular weight markers.

Solubility Studies--JhQ effects of pH were systematically examined using a

range of buffers at selected pH values, all prepared at a concentration of 10

mM. Samples of desmin (prepared from turkey gizzard smooth muscle

according to Huiatt et ai., 1980) and synemin, at a concentration of 0.2 mg/ml

each, were individually dialyzed extensively against a series of buffers at

different pH. The three buffers used were: 10 mM sodium-acetate, pH range

4.0-6.0; 10 mM imidazole-HCI, pH range 6.0-7.5; and 10 mM Tris-HCI, pH range 7.5-8.5. After dialysis, the samples were centrifuged at 183,000 x g for

1 h, the concentrations of protein in the supernatant were determined by using the modified Lowry procedure (Lowry et a!., 1951) supplied by the manufacturer (Sigma, Procedure No. P 5656), and data were expressed as percent of total protein sedimented. Aliquots of synemin obtained at selected pH values in the pH study (after dialysis, but before centrifugation) were negatively stained and examined by TEM as described earlier for purified synemin. The effects of ionic strength were systematically examined at both pH 7.0 and pH 8.5. Samples of desmin and synemin were individually dialyzed against Tris-HCI, pH 8,5, or against imidazole-HCI, pH 7.0, with NaCI added to 52 give selected ionic strengths. After dialysis for 16-18 h, the samples were

centrifuged either at high speed (183,000 x g) for 1 h or at low speed (17,000

X g) for 30 min, and the protein concentrations were measured and the results

expressed as described for the pH study.

Immunological /?ea5re/7fs--Rabbit polyclonal antibodies against avian synemin

were prepared essentially according to the methods of Knudsen (1985). The

electrophoretically-transferred synemin band was visualized by staining with

Ponceau S (Sigma) and a small strip of the nitrocellulose containing the 230

kDa synemin band was excised, washed extensively with glass-distilled HgO, dissolved in a minimum amount of dimethyl sulfoxide, and emulsified in an equal volume of complete Freund's adjuvant. Approximately 50 //g of protein was injected subcutaneously at multiple back sites of New Zealand White rabbits from which pre-immune serum had been collected and shown to test negatively by Western blot and immunofluorescence labeling analysis of skeletal muscle myofibrils. Starting 10 days after the first injection, the rabbits were boosted with ~50 //g of synemin in incomplete Freund's adjuvant at 14-20 day intervals. Rabbits were bled from an ear vein approximately 10 days after each boost. Monoclonal anti-desmin antibodies, D3, characterized by Western blot analysis (Danto and Fischman, 1984), were purchased from the Developmental

Studies Hybridoma Bank (Baltimore, MD). Monoclonal anti-a-actinin was purchased (Sigma). Polyclonal antibodies to porcine skeletal muscle desmin

(purified according to O'Shea et al., 1981) were prepared and characterized 53

essentially as described in Richardson et al. (1981). Monospecificity of the

anti-porcine skeletal muscle desmin, the anti-o-actinin, and the anti-synemin

antibodies also have been characterized elsewhere (S.R. Bilak et aL, 1991).

Western Blot Analysis--Sam^\es of intact chicken gizzard, chicken skeletal

muscle, and porcine smooth muscle were dissolved by homogenization in 10%

SDS, 0.1% MCE, 10 mM sodium phosphate, pH 7.0, and heating in a boiling

water bath for 5 min. After cooling, the samples were centrifuged at 2,000 x g

for 30 min, filtered, and aliquots of supernatant were used to prepare gel samples according to Laemmli (1970). Because synemin is a minor protein component of muscle (especially cardiac muscle), and because synemin copurifies with IF proteins (Granger and Lazarides, 1980), IF-enriched samples of chicken cardiac, porcine semitendinosus, and porcine cardiac muscles were prepared by the procedure of O'Shea et aL (1981). This included extraction of myofibril samples with 1 M Kl, 20 mM imidazole-HCI, pH 7.1, followed by one

HjO wash (corresponds to Step IX in O'Shea et aL, 1981), and subsequent solubilization of the resulting sediment in 6 M urea.

After separation by SDS-PAGE, proteins were transferred to nitrocellulose

(Bio-Rad) using a Bio-Rad transblot apparatus at 90 V for 1 h (using a Bio-Rad

Model 250/2.5 power supply) essentially as described by Towbin et aL (1979).

Protein transfer was confirmed by reversible staining with Ponceau S (Sigma).

The blots were blocked by incubation in Tris-buffered saline (TBS) (500 mM

NaCI, 20 mM Tris-HCI, pH 7.0) containing 2% Blotto (w/v) (non-fat, dry milk) 54 for 45 min with constant agitation, and then incubated with DEAE-Affi-Gel Blue

(Bio-Rad) affinity-purified polyclonal anti-synemin antibodies diluted 1:500 with

1 % Blotto (w/v) in TBS for 4-6 h at 25 °C with agitation. After three x 15 min

washes with 1 % Blotto (w/v) in TBS, the blots were incubated for 2 h at 25 "C

with horseradish peroxidase-goat-anti-rabbit IgG (Sigma) that had been diluted

1:1,000 with 1% Blotto (w/v) in TBS. After three x 15 min washes with 1%

Blotto (w/v) in TBS, the blots were developed using 4-chloro-1 -naphthol and

HjOj reagents for color reaction.

Myofibril and Tissue Preparations for lmmunocytochemistry--Myof\bx\\s were prepared from chicken thigh (mixed muscle)> chicken cardiac, porcine semitendinosus, and porcine cardiac muscles by procedures described elsewhere (M.M. Bilak et a!., 1991a). Intact tissue samples from adult chicken gizzard, skeletal (thigh) and cardiac muscles and from adult porcine stomach, semitendinosus, and cardiac muscles were removed promptly after death, cut into ~ 1 cm cubes, and immediately frozen in liquid nitrogen. The frozen blocks were mounted in OCT Compound Tissue-Tek freeze mounting medium (Miles

Scientific, Naperville, IL) on wooden blocks, 8 to 10 //m-thick sections were prepared by using an lEC-CTF microtome-cryostat, placed on acid-cleaned, gelatin-coated glass slides, and were stored at -70 °C.

Immunofluorescence Labeling-CxyosecWons were re-hydrated with phosphate-buffered saline (PBS) at 20 ®C followed by incubation with 5% bovine serum albumin (BSA) in PBS for 20 min at 20 °C to reduce non-specific 55 binding. Slides were washed three times with PBS at 4 "C, fixed with 0 °C

methanol for 5 min, washed three times with PBS at 4 "C, and then incubated

with primary antibodies for 30 min at 37 "C in a moist chamber. Polyclonal

anti-synemin and anti-desmin antibodies were diluted 1:50 with 1% goat serum

(Sigma) in PBS. Monoclonal antibody D3 was used in the form of undiluted

supernatants. After the primary antibody incubation, slides were washed three

times with PBS and then incubated with either fluorescein isothiocyanate- or

tetramethyl rhodamine isothiocyanate-labeled anti-rabbit IgG (for polyclonal

antibodies) or anti-mouse IgM (for monoclonal antibodies) (Cappel, West

Chester, PA) for 45 min at 20 ®C. Coverslips were fixed to the slides, and

samples were examined with a Zeiss III Photomicroscope equipped with

epifluorescence, using excitation filters for fluorescein or rhodamine.

Photographs were recorded on Kodak Tri-X or T-MAX 400 film. Controls included incubation of samples for 24 h at 4 "C with (1) pre-immune rabbit sera,

(2) 1 % goat serum in PBS, and (3) antibodies that had been pre-absorbed with the appropriate antigen. All controls demonstrated absence of specific labeling, and the background labeling was consistently low.

For immunofluorescent labeling of myofibrils, 1 to 2 ml of myofibril solution was washed three times with PBS to remove glycerol by centrifugation/ resuspension at 2,000 x p for 5 min (each). All the washing steps were done at 4 °C. Then 3 to 4 drops of the washed myofibril suspension were placed on to an acid-cleaned glass slide, spread with a coverslip, and allowed to settle. 56 Samples were then fixed with 0 °C methanol for 5 min, and immunolabeled as

described for the cryosections.

Confoca! Scanning Laser Microscopy (CSLM)"k Bio-Rad MRC-600 laser

scanning confocal microscope was used to examine co-distribution of desmin

and synemin in muscle cells. The samples were optically sectioned at 1 to 2

//m in the x-y direction. A series of confocal optical sections in the z direction

also were recorded to illustrate three-dimensional distribution of the antigens

throughout muscle myofibril bundles. Colocalization of double-labeled samples

was determined by pseudocolor overlaying of the digitized images. Images

were processed by a Northgate 33 MHz 386 computer and printed with a Sony

UP-5000 color video printer. All CSLM analyses were done at the Central

Electron Microscopy Research Facility, University of Iowa.

Image /Ina/ys/s-lmage analysis procedures were performed using a Zeiss

SEM-IPS image analysis system (IBAS 2000; Carl Zeiss, Germany). Images

were captured from 35mm negatives using a Sony 3 CCD color video camera.

For examining co-distribution of antigens in double-labeled samples, two

fluorescence images were superimposed to form a composite image. All image analysis procedures were done at the Image Analysis Facility, Iowa State

University.

Miscellaneous Methods-kwixno acid analyses were done on samples of chromatographically-purified synemin at: (1) the University of California, Davis, on a Beckman 6300 Amino Acid Analyzer using a ninhydrin based system. 57 Protein hydrolysis was done in vacuo at 110 "C for 24 or 72 h using 6 N HCI,

or for 24 h after performic acid treatment, and (2) the Protein Center, Iowa

State University, on an Applied Biosystems Amino Acid Analyzer using a

phenylisothiocyanate (PITC) based system. Samples were hydrolyzed in vacuo

in 6 N HCI at 150 °C for 1 h. Chromatographically-purified synemin also was

subjected to SDS-PAGE, and electrophoretically transfered to polyvinylidene

difluoride (PVDF) (Immobilon-P) membranes (Millipore Co., Bedford, MA) according to Matsudaira (1987). The 230 kDa synemin band was excised, hydrolyzed, and analyzed at the Protein Center, Iowa State University, as described above. All analyses were done in duplicate and data are expressed in mole percent. 58 RESULTS

Purification of Synemin--A synemin-containing, crude urea extract was prepared from turkey gizzard smooth muscle essentially as described by

Sandoval et ai. (1983). Because we were unable, in our hands, to routinely prepare highly homogenous synemin from the urea extract using their chromatographic protocol (Sandoval et a!., 1983), a different sequence of columns and elimination of phosphocellulose chromatography, was utilized herein. The elution profile obtained by hydroxyapatite chromatography of the crude urea extract is shown in Fig. 1A. The SDS-PAGE inset shows that the urea extract (U) contained some synemin (230 kDa), which migrates between the filamin (250 kDa) and myosin (205 kDa) standards (G), large amounts of contaminating desmin (53 kDa) and actin (42 kDa), and several minor components. Much of the actin did not bind to the column and was eluted in the first major peak {fractions A and B). Much of the desmin was eluted in the next major peaks between 20 and 40 mM phosphate (fractions C and D). A synemin-enriched fraction was eluted between 115 and 140 mM phosphate as a separate peak (fraction E). Additional purification was achieved by using a second hydroxyapatite (obtained from a different supplier) chromatography step. The column profile and the SDS-PAGE inset (Fig. IB) shows that more of the desmin and actin in the material loaded (S) was eluted as a broad initial peak (fraction A) and that synemin, containing less desmin and actin, was eluted between 120 and 140 mM phosphate (fraction B). Virtually complete FIG. 1. Chromatographic purification of synemin. A, Elution profile of the urea extract from a hydroxyapatite column. A total of 960 mg of protein in 480 ml of buffer B was loaded onto a 2.5 x 41 cm column and eluted with a phosphate gradient in buffer B (without Tris-HCI and PMSF) as indicated (dotted

Una). Flow rate was 11 ml/h and 5.5 ml fractions were collected. Inset shows SDS-PAGE analysis of gizzard standard (G), urea extract loaded (U), and the column fractions (A-E) pooled as indicated (vertical dashed Unes). Numbers to the left of the gel indicate approximate migration distances of filamin (250 kDa), myosin heavy chain (205 kDa), a-actinin (100 kDa), desmin (53 kDa), and actin (42 kDa). 3.5 250 GUA BC DE

3.0

^ 2.5 S G o 150 C C\2CO 2.0 (U u g k 100 o o Ui ^ 1.0

0.5

0.0

150 200 250 Tube Number FIG. 1 (cont'd).

B, Elution profile of hydroxyapatite-purified synemin (fraction E, Fig. 1A) from a second hydroxyapatite column. A total of 57 mg of protein in 110 ml of buffer B was loaded onto a 2.5 x 41 cm column and eluted with a phosphate gradient in buffer B (without Tris-HCI and PMSF) as indicated (dotted Une), Flow rate was 10 ml/h and 5.0 ml fractions were collected. Inset shows SDS-PAGE analysis of gizzard standard

(G), protein loaded (S = fraction E from first hydroxyapatite column), and the column fractions (A and B) pooled as indicated by the vertical dashed Unes. 0.8

100-

0 50 100 150 200 Tube Number 63 removal of the residual desmin and actin contaminants still present in the

synemin after two hydroxyapatite columns (see Fig. 1C inset, lane S) was

obtained by ion exchange chromatography on DEAE-Sephacel (Fig. 1C).

Purified synemin (fraction B) eluted between 135 and 160 mM NaCI. Every

fraction in the synemin peak was analyzed by SDS-PAGE. Western blot

analysis (results not shown) of the very initial portion of the peak routinely

contained some proteolytic breakdown products of synemin and, thus, was not included in the final "purified" synemin (Fig. 1C, fraction B). A total of 10.5 mg of purified synemin was obtained in the representative preparation shown in

Fig. 1. A composite SDS-PAGE analysis of the initial urea extract (U), synemin obtained from the first hydroxyapatite column (HI), synemin obtained from the second hydroxyapatite column (H2), and the final purified, pooled synemin from the DEAE-Sephacel column (S) is shown in Fig. 2A.

Selected properties of purified avian synemin--lhe amino acid analysis of purified gizzard synemin obtained by our procedure is shown in Table I and is compared with analyses reported by Sandoval et ai. (1983). Considering that analyses were done in different laboratories and with different methods, the amino acid composition of the synemins from our two laboratories was similar.

Electron micrographs of purified synemin, after removal of urea by dialysis against selected buffers, are shown in Fig. 3. In 10 mM Tris-HCI, pH 8.5, synemin exists primarily as spherical particles (10.8 ±0.15 nm; n = 200) by negative stain electron microscopy (Fig. 3A). Unidirectionally-shadowed FIG. 1 (cont'd).

C, Elutlon profile of hydroxyapatite-purified synemin {fraction B, Fig. 1B) from a DEAE-Sephacei column. A total of 23 mg of protein in 40 ml of buffer I was loaded onto a 1.6 x 33 cm DEAE-Sephacel column and eluted with a NaCI gradient in buffer I as indicated (dotted Une). Flow rate was 10 ml/h and 5.0 ml fractions were collected. Inset shows SDS-PAGE analysis of gizzard standard (G), protein loaded {S = fraction B from second hydroxyapatite column), and the column fractions (A and B) pooled as indicated by the vertical dashed lines. 1.0 250 G S A B G 250— ^ ^

100— ;f- 0.8

53—* 42-# - a A o 0.6 00 C\2 0) O ti 0} 0.4 k O m <

0.2

0.0 —1 I L_ 0 40 50 60 70 100 Tube Number FIG. 2. SDS-PAGE analysis of major fractions obtained during the purification of avian synemin and SDS-PAGE/Western blot analysis of fractions obtained during partial purification of porcine synemin. A, SDS-PAGE

composite analysis of avian synemin purification. Lane G = turkey gizzard standard with approximate migration distances of filamin (250 kDa), myosin heavy chain (205 kDa), a-actinin (100 kDa), desmin (53 kDa), and actin (43

kDa) marked at the left side; lane U = urea-extract loaded onto the first

hydroxyapatite column; lane HI = protein sample collected from the first column

(fraction E, Fig. 1A). Lane H2 = protein sample collected from the second

hydroxyapatite column (fraction B, Fig. IB) and loaded onto DEAE-Sephacel

column; lane S = DEAE-Sephacel purified synemin (fraction B, Fig. 1C). B, SDS- PAGE analysis of two fractions obtained from DEAE-Sephacel chromatography during partial purification of porcine stomach muscle synemin (see "Materials

and Methods"). Lane G = turkey gizzard standard; S = purified avian gizzard

synemin; F1 = fraction from the front portion of the porcine synemin peak

eluting from a DEAE-Sephacel column; F2= fraction from the back portion of the porcine synemin peak eluting from a DEAE-Sephacel column (see additional details in "Materials and Methods"). C, Western blot analysis of a duplicate gel to that shown in (B), using polyclonal antibodies to avian synemin. The 230 kDa synemin band is labeled in lane G (avian gizzard standard) and in iane S (purified avian gizzard synemin; a small amount of ~ 190 kDa degraded synemin also is evident). Bands at 225 kDa (prominent) and 195 kDa are present in F1 (porcine). These two bands also are present in F2 (porcine), but of more similar labeling intensity. A G u HI H2 s 68 TABLE I. Amino Acid Composition of Synemin in Mole Percent

Synemin Synemin Synemin Synemin Amino Acid chrom® chrom® PVDPC chrom" (SDS-PAGE)° Asp 9.1 9.8 9.3 8.5 9.3 Thr 7.3 6.7 7.1 7.3 6.7 Ser 9.3 9.8 10.0 11.1 10.5 Glu 19.2 18.5 19.6 20.7 20.1 Pro 2.1 2.7 2.4 ND ND Gly 5.3 6.8 7.5® 7.0 9.2® Ala 4.4 5.0 4.3 4.5 4.7 Val 6.7 5.6 6.8 6.7 6.4 Met 1.4 1.2 ND 1.1 1.0 lie 5.1 4.4 4.7 4.9 4.9 Leu 7.9 8.4 7.9 8.0 7.6 Tyr 2.2 2.2 2.6 2.5 2.4 Phe 2.8 2.9 2.6 2.6 2.9 Lys 7.3 7.3 6.5 7.0 6.1 His 2.9 2.3 2.1 • 2.6 2.6 Arg 6.9 7.4 6.5 6.3 5.5 Cys 0.4 ND ND 0.4 0.4

^Results from this lab using synemin purified by chromatography. Amino acid analysis was performed at the University of California, Davis (see "Materials and Methods"). ^Results from this lab using synemin purified by chromatography. Amino acid analysis was performed at Iowa State University (see "Materials and Methods"). '^Results from this lab using synemin additionally purified by preparative SDS-PAGE and polyvinylidine difluoride (PVDF) (see "Materials and Methods").

^Results from Sandoval eta!., (1983). Synemin (chrom) refers to their synemin purified by chromatography and synemin (SDS-PAGE) refers to their synemin purified by preparative SDS-PAGE. ®High value of glycine may be due to the buffer (Tris-glycine) used in preparative SDS- PAGE. FIG. 3. Electron micrographs of purified synemin. A, Negatively stained sample of synemin (0.1 mg/ml) after dialysis against 10 mM Tris-HCi, pH 8.5.

Synemin exists primarily as ~ 11 nm diameter spheres (arrow), and exhibits a slight tendency to self-associate (arrowheads). B, Unidirectionally-shadowed synemin (0.1 mg/ml) after dialysis against 10 mM Tris-HCI, pH 8.5. The synemin appears globular in form (arrow), and exhibits some tendency to self- associate (arrowhead). C, Negatively stained sample of synemin (0.1 mg/ml) after dialysis against 100 mM NaCI, 1 mM MgClj, 10 mM imidazole-HCI, pH 7.0. Under these conditions, where desmin forms IPs, synemin does not. Its appearance is less uniform than in A, existing in several shapes, including extended (arrow), and approximately spherical (arrowhead). The latter particles often show evidence of self-association (arrowhead). D, Negatively stained sample of synemin (0.1 mg/ml) that had first been dialyzed extensively against 4 M guanidine-thiocyanate, 0.2 M NaCI, 1 mM DTT, 0.5 mM EGTA, 100 mM MES, pH 6.5, followed by extensive dialysis against 0.2 M NaCI, 1 mM DTT, 0.5 mM EGTA, 100 mM MES, pH 6.5. Under these conditions, where neurofilament-M forms short filaments (Gardner ef a/., 1984), synemin forms similar extended particles (arrow). Bars = 0.^ //m. mm 71 samples of synemin in the same buffer (10 mM Tris-HCI, pH 8.5) also

demonstrated the spherical nature of synemin (Fig. 3B). Negatively stained

samples of synemin, under conditions in which purified desmin forms 10-nm

filaments (Huiatt et a!., 1980; Chou et a!., 1990), show that the synemin

molecules are less uniform in appearance, have a marked tendency to self-

associate, but are not filamentous (Fig. 3C). Examination of negatively-stained

synemin, after dialysis against 130 mM KCI, 0.1 mM dithiothreitol (DTT), 20

mM Tris, pH 7.4 ("buffer E"), the same buffer used in studies by Sandoval et al.

(1983), gave appearances similar to that shown in Fig. 3C (results not shown).

Dialyzing synemin extensively against solutions in which NF-M self-assembles

into IF-like 10-nm filaments (Gardner ef a/., 1984; Mulligan eta!., 1991) causes

synemin to form short, ~S-shaped structures (~10 nm in diameter) (Fig. 3D),

but not the typical long 10-nm filaments formed from desmin (Huiatt et at.,

1980).

The results of crosslinking purified synemin in low ionic strength, slightly

alkaline solutions with glutaraldehyde are shown in Fig. 4 (lanes 1-5). The effect of time (0-30 min) on crosslinking soluble synemin at constant protein concentration (0.1 mg/ml) in 10 mM triethanolamine-HCI, pH 8.5, indicated that an intramolecularly crosslinked species, which corresponds in molecular mass to about 460 kDa (presumably a dimer), becomes the principal product (Fig. 4A cf., lanes 1 and 2 with lane 4). Doubling the glutaraldehyde concentration to

0.04% glutaraldehyde per 0.05 mg protein/ml (synemin still present at 0.1 FIG. 4. Chemical crosslinking analysis of purified synemin. A, Effect of time and ionic strength on giutaraidehyde crosslinking of synemin as shown by SDS-

PAGE (5% acrylamide). Lanes 1-5, synemin (0.1 mg/ml) crosslinked in 10 mM

triethanolamine-HCI, pH 8.5. Lane 1 =0 time (control); lane 2 = 1 min; lane

3 = 5 min; lane 4 = 30 min; lane 5 = 5 min, but with a giutaraidehyde concentration (giutaraidehyde = 0.04%/0.05 mg/ml synemin) double that used

in lanes 1-4 (giutaraidehyde =0.02%/0.05 mg/ml synemin). Lanes 6-8, synemin (0.1 mg/ml) crosslinked (giutaraidehyde =0.02%/0.05 mg/ml synemin)

in 65 mM KCI, 0.1 mM DTT, 10 mM triethanolamine-HCI, pH 8.5. Lane 6 = 1

min; lane 7 = 5 min; lane 8 = 30 min. Lane 9= turkey gizzard standard and the

numbers to the right indicate the approximate migration distances of

polypeptides having the indicated M, x 10®. Numbers to the left indicate the migration distance of synemin (230 kDa) and crosslinked synemin (-460 kDa). B, Effect of protein concentration on giutaraidehyde crosslinking of synemin as

shown by SDS-PAGE (2% acrylamide, 3% agarose). Lane 1 = crosslinked

phosphorylase standards and the numbers to the left indicate the approximate migration distances of monomer (97 kDa), dimer (195 kDa), trimer (292 kDa),

tetramer (390 kDa), and pentamer (487 kDa); lane 2= turkey gizzard standard

and the asterisk corresponds to the migration distance of the myosin heavy

chain (205 kDa); lane 3 = 0.1 mg/ml synemin; lanes A and 6 = 0.2 mg/ml

synemin; ianes 5 and 7 = 0.5 mg/ml synemin; iane 8=0 time (control). Lanes 3-5 contained 0.02% glutaraldehyde/0.05 mg/ml synemin during crosslinking,

and ianes 6 and 7 contained 0.04% glutaraldehyde/0.05 mg/ml synemin during

crosslinking. Lane 8=0 time (control). Numbers to the right indicate the approximate migration distances for synemin (230 kDa), crosslinked synemin dimer (460 kDa), and putative crosslinked synemin tetramer (920 kDa). The arrowhead= approximate migration position of a trace of putative trimer (690 kDa). 1 23456789 g12345678 74

mg/ml) showed a similar crosslinking pattern (Fig. 4A, lane 5; compare with

lane 3), indicating that the amount of giutaraldehyde was sufficient, increase in

ionic strength by addition of salt, however, changed the crosslinking pattern

(Fig. 4, lanes 6-8). Crosslinking for only one minute, but in the presence of 65

mM KCI (Fig. 4, lane 6), yielded a very large crosslinked product that did not

enter the 5% acrylamide gel, although a small amount of uncrosslinked synemin

monomer (230 kDa) and crosslinked dimer (460 kDa) was still evident. With

increasing time of crosslinking, the amount of these two latter species was

reduced (Fig. 4A, lanes 7 and 8). The effect of synemin concentration on

crosslinking of soluble synemin in 10 mM triethanolamine-HCI, pH 8.5, with a

constant ratio of giutaraldehyde (0.02% giutaraldehyde per 0.05 mg protein/ml)

is shown in Fig. 4B (lanes 3-5). The dimer (-460 kDa) is the predominant

species, and remains so as protein concentration increases (Fig. 4B, cf., lanes

3-5), indicating the soluble synemin molecule contains two 230 kDa polypeptides. No change in the crosslinking pattern is observed by doubling the amount of giutaraldehyde to 0.04% giutaraldehyde per 0.05 mg protein/ml (Fig.

4B, lanes 6 and 7), indicating that the amount of giutaraldehyde was sufficient.

An approximately 920 kDa species, possibly a tetramer, which increases slightly in amount with increase in synemin concentration (Fig. 48, lanes 3-5) may be an intermolecularly crosslinked product of synemin.

Two series of experiments were done to examine and compare the effects of pH and ionic strength on solubility as measured by sedimentation of desmin and 75 synemin. Soluble desmin present in 10 mM Tris-HCI, pH 8.5, self-assembles

into long, approximately 10-nm diameter filaments after dialysis against

solutions of lower pH (<8.0) or increasing ionic strength (Huiatt et a!., 1980;

Hartzer, 1984; Ip et a/., 1985a; Chou et al., 1990; and, results not shown). By

plotting the percentage of protein sedimented, a measure of the yield of

filaments or of non-filamentous aggregates is achieved (Huiatt, 1979). The

effect of pH (at a constant low ionic strength) on desmin and synemin is shown

in Fig. 5. Each protein (0.2 mg/ml) was individually dialyzed against 10 mM

buffers at selected pH values, centrifuged, and the amount of protein remaining

in the supernatant was measured. The percentage of desmin sedimented

increased (solubility of desmin decreases) as the pH was lowered below pH 8.5.

Approximately 50% of the desmin was sedimented at pH 6.8, and this

percentage increased to about 98% at pH 6.5. The percentage of synemin

sedimented increased (solubility of synemin decreases) as the pH was lowered

below pH 7.75. Approximately 50% of the synemin sedimented at pH 6.4, and

this percentage increased to about 90% at pH 6.1. Both proteins continue to

be nearly completely sédimented as pH is lowered to a pH of ~4.0, and then

both proteins became more soluble with further decrease in pH (Fig. 5).

Overall, the shapes of the two pH curves are similar, with the synemin curve

shifted about one-half pH unit lower. Samples of synemin were selected from

throughout the pH range (before sedimentation), negatively stained, and examined by electron microscopy. Representative micrographs at four pH FIG. 5. Effect of pH at low Ionic strength on amount of purified desmin and synemin sedimented. Samples of purified desmin or synemin at 0.2 mg/ml were dialyzed individually against 10 mM buffers as described in the "Materials and Methods." Buffers used were: pH 4.0-6.0, 10 mM sodium-acetate; pH 6.0-7.5, 10 mM imidazole-HCI; pH 7.5-8.5, 10 mM Tris-HCI. Samples were centrifuged at 183,000 x y for 1 h and the protein remaining in the supernatant was determined by using the modified Lowry procedure (Sigma). Data are from three separate preparations of each protein run in triplicate. PERCENTAGE OF TOTAL PROTEIN SEDIMENTED

O M

00 78 values are shown in Fig. 6. Unlike desmin, which forms filaments as pH is

lowered to pH -7.00 (Huiatt et a!., 1980), most of the synemin at pH 7.27

(Fig. 6A) remains as dispersed, approximately spherical particles of -11 nm

diameter, with a small number of larger aggregated (or associated) structures

evident. At pH 6.63 (Fig. 6B), a large increase in the amount of larger,

aggregated particles is demonstrated. The synemin demonstrated a marked

propensity to form large, aggregated complexes or networks in the pH range of

6 to 4 (e.g., pH 4.95 in Fig. 6C). With further decrease in pH, synemin formed

less compact aggregated networks (e.g., pH 3.95 in Fig. 6D).

The effect of ionic strength on the amount of synemin sedimented was

examined in a manner analogous to the pH study. Samples of either synemin or

desmin (for comparison) were individually dialyzed against Tris-HCI, pH 8.5, or

imIdazole-HCI, pH 7.0, buffers of Increasing ionic strength (adjusted with NaCI),

were centrifuged either at high or low speed, and the amount of protein sedimented was recorded (Fig. 7). Because desmin forms filaments at low pH values, even at very low ionic strength, and, thus, essentially all would be sedimented with high speed centrifugation, only low speed centrifugation was performed on the samples studied at pH 7.0. For 50% sedimentation with low speed centrifugation at pH 7.0, synemin requires a higher ionic strength (0.08) than does desmin (0.03) (Fig. 7, circles). With increasing ionic strength at pH

7.0, more desmin {circles) continues to sediment (becomes less soluble), whereas the amount of synemin sedimented levels off and remains at ~ 50% FIG. 6. Electron micrographs of synemin at selected pH values. Small aliquots of synemin, which had been dialyzed against low ionic strength buffers at selected pH values and sampled before sedimentation (see Fig. 5), were negatively stained and examined by electron microscopy. A = pH 7.27. Most of the synemin is still dispersed and exists as -11 nm diameter spheres

(arrow), however the synemin has a tendency to aggregate as the pH is lowered from pH 8.5 and a few larger aggregates are observed (arrowhead).

B = pH 6.63. The number of larger aggregates (arrowhead) observed has significantly increased. C = pH 4.95. At this pH synemin appears as a highly aggregated complex. D = pH 3.95. With further decrease in pH, synemin is less complexed, but is still fairly aggregated. flar=0.25 //m.

FIG. 7. Effect of Ionic strength on amount of purified desmin and synemin sedimented. Samples of purified desmin (top graph) and synemin (bottom graph) at 0.2 mg/ml were individually dialyzed against a series of solutions of increasing NaCI concentration buffered either at pH 7.0 with 10 mM imidazole- HCI or at pH 8.5 with 10 mM Tris-HCI. Samples were centrifuged at either low

(17,000 X g for 30 min) or high (183,000 x gr for 1 h) speed, and the amount of protein remaining in the supernatant was measured by using the modified Lowry procedure (Sigma). Data are from two separate preparations of each protein run in triplicate. Sfyt/ares = high speed centrifugation, pH 8.5; circles = \ovj speed centrifugation, pH 7.0, and f/va/jfl'/es = low speed centrifugation, pH 8.5. PERCENTAGE OF TOTAL PROTEIN SEDIMENTED PERCENTAGE OF TOTAL PROTEIN SEDIMENTED

ro 4k O) 00 O oo o O O O O 83 between ionic strengths of 0.08 and 0.16. At pH 8.5, where both proteins are

essentlaliy completely soluble (not sedimented at either centrifugation speeds),

significant amounts of both synemin and desmin are sedimented with high

speed centrifugation (Fig. 7, squares) as ionic strength increased slightly. At

ionic strengths greater than 0.04, ~90% of desmin and ~75% of synemin are

sedimented {squares). At pH 8.5, increasing amounts of desmin sediments

with low speed centrifugation as the ionic strength increases, whereas the

synemin sedimented increases, but levels off at ~25% between 0.08 and 0.16

(Fig. 7, triangles). Huiatt (1979) has shown that, in the case of desmin, this

increase in amount sedimented is a result of ionic strength on IF elongation.

The shape of the solubility/sedimentation curves obtained with synemin by

using low speed centrifugation were biphasic at pH 7.0 (circles) and at pH 8.5

(triangles). SDS-PAGE analyses of all supernatants and pellets after

centrifugation (from both the pH and the ionic strength studies) showed that the relative amounts of protein present in the supernatants and pellets corresponded to the data points shown in Figs. 6 and 7, and indicated no difference in their composition (results not shown).

Western Biot Analysis--\Nesterx\ blot analysis of fractions prepared from adult chicken and porcine smooth, skeletal, and cardiac muscles shows that polyclonal antibodies to turkey gizzard synemin labels reactive antigens in all six samples (Fig. 8). In all three chicken muscle fractions (Fig. 8B, lanes 2-4), the anti-synemin antibodies recognize the 230 kDa synemin band. The antibodies FIG. 8. SDS-PAGE and Western blot analysis of adult chicken and porcine muscle fractions with rabbit antibodies to avian gizzard synemin. Adult avian and porcine smooth, skeletal, and cardiac muscle fractions were separated on 8.5% SDS-poIyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. A, SDS-polyacrylamide gel stained with Coomassie Blue; B, Western blot of a duplicate gel shown in (A) incubated with anti-

synemin. Lane 1 = purified turkey smooth (gizzard) synemin; lane 2 = whole

homogenate of chicken smooth (gizzard) muscle; lane 3 = whole homogenate of

chicken skeletal muscle; lane 4 = IF-enriched fraction from chicken cardiac

muscle; lane 5 = whole homogenate of porcine smooth (stomach) muscle; lane

6 = IF-enriched fraction from porcine skeletal muscle; and lane 7 = IF-enriched fraction from porcine cardiac muscle. Samples were prepared as described in the "Materials and Methods." As shown in (B), anti-synemin antibodies

recognize the 230 kDa synemin in the avian synemin control (lane 1 ) and in all

three of the avian muscle fractions (lanes 2-4). A small number of bands, including one at ~ 190-200 kDa, that all increase in amount during extended

storage, also are evident in the avian smooth muscle sample (lane 2). Two bands corresponding to migration distances of proteins at ~ 225 kDa and ~ 195 kDa (labeled at right) axe present in all three of the porcine muscle fractions

(lanes 5-7). The desmin present in the porcine muscle fractions (A, lanes 5-7) has a slightly slower mobility (55 kDa) than that of the avian desmin (53 kDa) when separated by SDS-PAGE run by the Laemmli (1970) procedure as we have previously shown (O'Shea et aL, 1979). Positions of filamin (250 kDa), purified gizzard synemin (230 kDa), myosin heavy chain (205 kDa), a-actinin

(100 kDa), desmin (53 kDa), and actin (42 kDa) are shown at the left of A.

86 to avian synemin recognize two bands, corresponding in migration positions to

proteins with molecular masses of ~225 kDa and ~ 195 kDa, in all three

porcine muscle fractions (Fig. 8B, lanes 5-7).

To provide additional evidence that the two bands labeled in the porcine muscle fractions were the mammalian synemin homolog(s) of avian synemin, synemin was partially purified from porcine stomach smooth muscle (Fig. 2, B and C). A peak corresponding in elution position to that obtained with avian synemin (Fig. 1 A, peak E) was obtained by chromatographing the crude urea extract prepared from porcine stomach smooth muscle on a hydroxyapatite

(Bio-Rad) column (results not shown). The peak contained, in addition to remaining desmin and actin contaminants, proteins of -225 kDa and -195 kDa. When the fractions in this peak were pooled and chromatographed on a

DEAE-Sephacel column, a peak corresponding in elution position to that obtained with avian synemin by DEAE-Sephacel chromatography (Fig. 1C, peak

B) was obtained (results not shown). Analysis of the front (lane F1) and back

(lane F2) parts of the peak by SDS-PAGE (Fig. 2B) showed that the peak was considerably enriched in the -225 kDa and -195 kDa proteins. Western blot analysis of these fractions indicated that these two bands were recognized by antibodies to avian synemin (Fig. 2C), showing that the -225 kDa and -195 kDa proteins represent the mammalian homolog(s) of avian synemin.

Immunofluorescence localization of synemin-Jhe localization of synemin (or homolog) was examined in adult chicken and porcine smooth, skeletal, and 87 cardiac muscles by indirect immunofluorescence microscopy. Myofibrils or

frozen tissue samples were first treated with specific primary antibodies

followed by incubation with an appropriate secondary antibody. Paired phase

and immunofluorescence images of anti-a-actinin, anti-desmin, and anti-

synemin-labeled myofibrils from chicken thigh muscle are shown in Fig. 9, A-F.

Myofibrils labeled with anti-a-actinin (Fig, 9, A and B) show the characteristic

solid (continuous) Z-line staining pattern danger and Pepe, 1980). Myofibrils

labeled with anti-desmin (Fig. 9, C and D) and anti-synemin (Fig. 9, E and F) both show Z-line staining patterns that are punctate in nature, as is characteristic for anti-desmin labeling (Richardson et a/., 1981). Chicken skeletal muscle myofibrils that were double-labeled with anti-desmin and anti- synemin show that both antibodies decorate the myofibrillar Z-lines (Fig. 9, G-l).

Superimposing the photographic negatives of the phase contrast (Fig. 9G) and the two fluorescence images (Fig. 9, H and I) of double-labeled myofibrils with the aid of image analysis (Fig. 9J) shows that the labeling patterns obtained with antibodies to desmin and synemin coincide and are at the Z-lines. A fragment of a chicken skeletal muscle myofibril bundle that was stained with anti-synemin and optically sectioned using a confocal microscope is shown in

Fig. 10. Longitudinal sections in the z direction near the top, middle, and bottom (Fig. 10, B-D) of the bundle fragment shown in Fig. 10A (phase image) shows that the anti-synemin antibodies stain throughout the myofibril bundle.

In Fig. ICE, a chicken muscle myofibril fragment (inset shows the phase image) FIG. 9. Indirect immunofluorescence microscopy of adult chicken skeletal muscle myofibrils. (A-F), Paired phase contrast (A, C, and E) and immunofluorescence (B, D, and F) micrographs of chicken skeletal muscle myofibrils labeled with monoclonal anti-a-actinin (A and B), polyclonal anti- desmin (C and D), and polyclonal anti-synemin (E and F) antibodies. (A and B), ff-Actinin antibodies show the characteristic continuous staining pattern on the myofibrillar Z-lines. (C and D), Anti-desmin antibodies show the characteristic punctate staining pattern at the myofibrillar Z-lines. (E and F), Anti-synemin antibodies stain the Z-lines in a punctate pattern similar to that of anti-desmin.

Bar in F is for A-F = 10 yt/m. (G-l), Myofibrils double-labeled with monoclonal anti-desmin (D3 of Danto and Fischman, 1984) and polyclonal anti-synemin. Phase contrast (G) and immunofluorescence labeling of the same myofibril show the staining patterns with both anti-desmin (H) and with anti-synemin (I) were similar, i.e., they both specifically label the Z-lines. (J), A computer generated image from the three photographic negatives of the labeled myofibril fragment shown in G-l demonstrates that the labeling locations with anti-desmin and anti- synemin coincide at the Z-line. Bar in J is for G-J = 10 /vm. — , • \ r ' I, ) •• A, STriËiEa^ I iill i m jI •

H

ill'. AI g \11 .liii'.j i' . i.i I< !11 nil I , FIG. 10. CSLM of adult chicken skeletal muscle myofibrils stained with anti- synemin. A, Phase contrast image of the chicken myofibril bundle/fragment.

Bar=^0 fjn\. (B-D), Confocal optical sections taken near the top (B), middle (C), and bottom (E) of the chicken myofibril fragment show that the anti- synemin staining is present throughout the myofibril bundle/fragment. Bar-'XQ //m. E, A fluorescence micrograph of a chicken myofibril fragment (//7sef = phase contrast image). The periodicity of anti-synemin antibodies is shown by the scan Une graph. The fluorescence intensity signals coincide with the myofibrillar Z-lines. The horizontal line between the two x's {white open arrows) represent the entire scan line and includes 13 Z-lines. For a reference position, the verticaiUne on the scan corresponds to the seventh Z-line

(arrowhead). Distance on the abscissa pertains only to the scan line. Bar='[0 /im, and pertains only to the myofibril. 0 5 10 15 20 25 30 DISTANCE BETWEEN LINES (microns) 92 was optically scanned using the confocal microscope in the x-y plane for the

distance spanned by thirteen sarcomeres (the two ends of the distance scanned

is shown by the white open arrows). The periodicity and relative fluorescence

intensity were plotted and are shown (Fig 10E). The vertical line represents a

reference point, which was placed on the seventh Z-line from the left edge of

the distance scanned, showing that the peak of the fluorescence intensity

coincides with the myofibrillar Z-lines. The distance between the peaks of the

fluorescence intensity is ~2.5 //m, which matches the sarcomere length of the

myofibrils shown in Fig 10E.

In Fig. 11, A-C, chicken cardiac muscle myofibrils (Fig. 11 A, phase contrast)

that were double-labeled with anti-desmin (Fig. 11B) and anti-synemin (Fig.

11C) show colocalization of antibodies at the cardiac myofibrillar Z-lines.

Cryosections of chicken cardiac muscle that were stained with anti-synemin

show a specific sarcomeric labeling pattern (Fig. 11, D and E). When

cryosections of chicken cardiac muscle were incubated with pre-immune serum

obtained from the rabbit used for production of anti-synemin antibodies, background labeling is insignificant (Fig. 11, F and G), which indicates that the staining pattern shown in Fig. 11 (A-E) is specific.

To further investigate and demonstrate the presence of synemin (or a homolog) inside adult mammalian muscle cells, both isolated myofibrils from, and frozen sections of, adult porcine skeletal, smooth, and cardiac muscle were probed with antibodies to avian synemin. Fig. 12 shows a series of porcine FIG. 11. Indirect immunofluorescence localization of synemin in adult chicken cardiac muscle samples. (A-C), Myofibrils double-labeled with monoclonal anti-desmin (D3 of Danto and Fischman, 1984) and polyclonal anti- synemin. Phase contrast (A) and immunofluorescence images of the same myofibrils show that both desmin (B) and synemin (C) antibodies specifically stain the myofibrillar Z-lines {arrows). Bar=^0 fjm. (D-G), Cryosections of adult chicken cardiac muscle. (D and E), Paired phase contrast (D) and immunofluorescence (E) micrographs of samples stained with anti-synemin showing a periodic sarcomeric staining pattern. (F and G), Paired phase contrast (F) and immunofluorescence (G) micrographs of samples stained with pre-immune serum from the same rabbit (before immunization) that produced the anti-synemin antibodies used in A and C and in D and E show that background labeling is negligible. Bar in G is for D-G = 10 //m.

FIG. 12. Indirect immunofluorescence microscopy and CSLM of adult porcine skeletal muscle samples. (A and B), Paired phase contrast (A) and indirect immunofluorescence (B) micrographs of porcine skeletal muscle myofibrils labeled with polyclonal anti-avian-synemin show a punctate labeling pattern at the Z-lines (arrows). (C-E), Myofibrils double-labeled with monoclonal anti-desmin (D3 of Danto and Fischman, 1984) and polyclonal anti-synemin. Phase contrast (C) and indirect immunofluorescence labeling show that the staining patterns with both the anti-desmin (D) and with anti-synemin (E) were very similar, i.e., they both label the Z-lines [arrows). (F and G), Confocal optical sections of a myofibril fragment in the x-y plane that was double-labeled with monoclonal anti-desmin (F) and polyclonal anti-synemin (G). The computer derived composite (H) of F and G shows that the labeling positions of the two antibodies at the Z-line (arrows) coincide. (I and J), Cryosections of adult porcine semitendinosus muscle. Paired phase contrast (I) and immunofluorescence (J) micrographs of samples labeled with anti-synemin show that the anti-synemin antibodies decorate Z-lines. Arrows point to the Z- lines in all cases. Bar-^0 /jm. f,kfwW4, 97 skeletal muscle myofibrils that were labeled with anti-synemin and/or desmin

antibodies. Fig. 12, A and B are paired phase (Fig. 12A) and fluorescence (Fig.

12B) micrographs of anti-synemin-labeled myofibrils. The presence of synemin at the Z-lines is evident. To demonstrate colocalization of synemin and desmin, the myofibrils were, in some cases, also labeled with anti-desmin (Fig. 12, C-H).

Double-labeled porcine siceletal muscle myofibrils (Fig. 12, C-E) show that the staining pattern obtained with anti-synemin (Fig. 12E) is similar to that of anti- desmin (Fig. 12D); i.e., both antibodies label the Z-lines in a punctate pattern.

Another example of porcine skeletal muscle myofibrils (a myofibril fragment/bundle), that was double-labeled with anti-desmin and anti-synemin is shown in Fig. 12, F and G, respectively. Superimposing these two images shows that the antibody staining patterns are in exact register and coincide at the Z-line (Fig. 12H). Anti-synemin-labeled frozen sections of porcine semitendinosus muscle (Fig. 12, I and J) demonstrate a periodic labeling pattern, and when similar cryosections are sectioned in the x-z plane by confocal microscopy, the presence of synemin is found at the Z-line level throughout the sections (results not shown).

Examination of anti-avian-synemin labeling in adult porcine cardiac muscle samples is shown in Fig. 13. Cardiac muscle myofibrils double-labeled with anti-desmin and anti-synemin are shown in Fig. 13, A-C. Immunofluorescence micrographs double-labeled with anti-desmin (Fig. 138) and anti-synemin (Fig.

13C), and the corresponding phase contrast micrograph (Fig. 13A), show that FIG. 13. Indirect immunofluorescence localization of synemin in adult porcine cardiac muscle samples. (A-C), Myofibrils double-labeled with monoclonal anti-desmin (D3 of Danto and Fischman, 1984) and polyclonal anti- synemin (avian). Phase contrast (A) and immunofluorescence labeling of the same myofibril fragment show that the staining patterns with both anti-desmin (B) and anti-synemin (C) were similar and that they both stain the Z-lines

(arrows). (D-F), Cryosections of adult porcine cardiac muscle. (D and E), Paired phase contrast (D) and immunofluorescence (E) micrographs of samples stained with anti-synemin show a periodic sarcomeric staining pattern. F, Immunofluorescence micrograph of a cryosection of adult porcine cardiac muscle treated with anti-synemin showing that the intercalated disk [arrow), as well as the Z-lines, are labeled with anti-synemin antibodies. Bars=^0

100 both antibodies label the cardiac Z-lines. Fig. 13, D and E are paired phase (Fig.

13D) and immunofluorescence (Fig. 13E) micrographs of cryosections of cardiac muscle that were stained with anti-synemin antibodies, and demonstrate a sarcomeric staining pattern with anti-synemin. As shown in the immunofluorescence micrograph of a cardiac muscle cryosection including intercalated disks (Fig. 13F), anti-synemin stains the intercalated disk region in addition to the Z-lines.

Indirect immunofluorescence labeling of adult porcine stomach smooth muscle cryosections that were labeled with anti-synemin shows that anti- synemin labeling is observed in a wavy pattern throughout the smooth muscle cells (Fig. 14). FIG. 14. Indirect immunofluorescence localization of synemin in cryosections of porcine smooth muscle. (A and B), Paired phase contrast (A) and immunofluorescence (B) micrographs of a longitudinal section that was incubated with anti-synemin show the presence of synemin (or homolog) in mammalian smooth muscle. Bar=^0 ijm.

103 DISCUSSION

Insofar as we are aware, all previous studies on synemin have originated from but one laboratory (Granger and Lazarides, 1980, 1982, 1984; Granger et a!., 1982; Lazarides et at., 1982; Price and Lazarides, 1983; Sandoval et a!.,

1983), and Sandoval et al. (1983) reported a procedure for preparation of synemin. We have utilized much of this procedure, especially for isolation of the crude urea extract. But, we have been largely unsuccessful in routinely preparing satisfactorily homogeneous and nondegraded synemin in reasonable quantities following their chromatographic protocols. Comparing in detail our purification studies to theirs is difficult because they did not show chromatographic profiles or report yields (Sandoval et a/., 1983). The significant changes devised herein which, at least in our hands, results in a more reliable scheme, include: (1) addition of more protease inhibitors/bacteriostats during preparation of the acetone powder, because synemin is extremely sensitive to proteolysis (Granger and Lazarides, 1980;

Sandoval et a!., 1983; and observed herein); (2) using repeated hydroxyapatite chromatography to remove nearly all of the two major contaminants, desmin and actin, present in the crude 6 M urea extract obtained from the acetone powder. Hydroxyapatite has been shown to be especially useful for removal of actin from other cytoskeletal proteins (e.g., Suzuki et aL, 1976; Huiatt et a!.,

1980). Using two commercial hydroxyapatites, with different properties (see

"Materials and Methods"), in the order described herein, and avoiding 104

overloading of the first column with protein as described earlier {Sandoval et a!.,

1983), proved valuable; and (3) elimination of phosphocellulose

chromatography. Synemin resulting from the second hydroxyapatite column

(see Fig. IB, fraction B) was of sufficient quality that it could be purified to

satisfactory homogeneity by DEAE-Sephacel chromatography without the one

or two phosphocellulose chromatography steps previously described as

necessary (Sandoval et a/., 1983). We found the results of phosphocellulose

chromatography to be highly variable, often leading to significant protein loss,

additional proteolysis, and poor separation of contaminants. Using the procedures described herein, our yields of purified synemin were in the range of

9 to 15 mg protein from nine separate preparations.

Selected Properties of S/nemm- Removal of urea from chromatographically- purified synemin by dialysis against 10 mM Tris-HCI, pH 8.5, results in a soluble protein that appears spherical (diameter = -11 nm) in negatively stained images. Chemical crosslinking by using either glutaraldehyde (Fig. 4), or ethylene alvcolbis(succinimidyl succinate) (results not shown), indicated that synemin molecules in low ionic strength solutions at pH 8.5 contained two 230- kDa polypeptides. Although it was reported that synemin has solubility properties similar to those of desmin and vimentin (Granger and Lazarides,

1980), that conclusion was based upon copurification of proteins during initial steps of preparation, and on the observation that immunofluorescent localization of synemin on myofibrils and Z-line sheet structures was not altered 105 (removed) by treatment of such specimens with non-ionic detergent or

detergent and high salt, which also was the result obtained with localization of

the IF proteins. Comparison of the solubility of purified synemin with that of

purified IF proteins has not been previously reported. As shown in our studies

(Figs. 5 and 6), synemin and desmin do share some general similarity in their

pH- and ionic strength-dependent solubility properties, but there are some significant differences as well. Examination of negatively stained samples showed that under conditions in which desmin or vimentin tetrameric protofilaments assemble into IFs (Huiatt et a!., 1980; Ip et a!., 1985a,b; Chou et a!., 1990), synemin does not. Instead, as the pH is lowered into the physiological range (see Fig. 6A, B) and/or ionic strength is increased (see Fig.

3C), synemin forms overall globular-lilte, but less regular structures of ~ 15-25 nm diameter. Scrutiny of these complex particles (Fig. 3C, Fig. 6B) indicates they are simply aggregates of the ~ 11 nm diameter synemin molecules, and resemble the somewhat larger ( - 35-40 nm diameter) globular-like structures observed for rotary shadowed images of synemin shown by Sandoval et a/.,

(1983).

Sandoval et at. (1983) reported that synemin in 130 mM KCI, 0.1 mM dithiothreitol, 20 mM Tris, pH 7.4 (buffer E) had a molecular weight of

980,000, as determined by gel filtration, and that synemin is a tetramer. We were unable to confirm their conclusion that the soluble form of synemin is a tetramer. Attempts to chemically crosslink synemin in solutions similar or 106 identical to theirs yielded large crosslinked products that failed to enter 5%

polyacrylamide gels (30:0.6% acrylamide:bisacrylamide), or that migrated as a

heterogeneous smear near the top of very porous 2% polyacrylamide/3%

agarose gels. In view of the considerably heterogeneous nature described for

the synemin in Sandoval et a!., (1983), our aforementioned crosslinking results, and the presence of aggregates comprised of ~ three to five of the 11 nm diameter spheres we observed in negatively stained images of synemin in their buffer E, we feel it is unlikely that the isolated soluble synemin rnolecule exists as a tetramer. Our solubility and TEM experiments of synemin, conducted over a range of pH and ionic strength, demonstrated that synemin has a rnarked propensity to aggregate into globular-like particles of increasing size and complexity. It also was evident that purified synemin would not assemble into typical IPs under conditions in which purified desmin assembles into long IPs.

The similarity. If any, of synemin to other IPAPs, or to any of the IP proteins, is not clear. Synemin is sometimes grouped into a class of IPAPs considered to have properties or roles as crosslinking components (i.e., crosslinking IPAPs)

(Steinert and Roop, 1988). One of the members of this IPAP class is paranemin, a poorly characterized ~280 kDa protein, present in all developing avian myogenic cells and in adult avian cardiac muscle cells (Breckler and

Lazarides, 1982). No antibody cross-reactivity between avian synemin and paranemin has been observed (Breckler and Lazarides, 1982; Price and

Lazarides, 1983). Paranemin has not been purified and characterized; thus, few 107 other properties/characteristics of the protein are known.

Plectin, another member of the crosslinking IFAP class (Steinert and Roop,

1988; Robson, 1989; Foisner and Wiche, 1991), has been studied in

considerable detail (Wiche et aL, 1991). Although also present in muscle cells,

plectin, unlike synemin, is present in a wide variety of cell types (Wiche, 1989).

Contrary to synemin, plectin molecules are long, dumbbell-like structures

(Foisner and Wiche, 1987; Foisner et a!., 1991), and chemical crosslinking

studies indicate plectin is a tetramer in low ionic strength, slightly alkaline

solutions (Foisner and Wiche, 1987). Thus, it seems likely that plectin and

synemin are distinct proteins, although they may be shown to be functionally

related.

Mammalian neurofilaments are heteropolymers of three polypeptides known

as the neurofilament triplet, NF-L, NF-M, and NF-H (Liem, 1990). Although all

three NF peptides are considered IF proteins because they contain the

conserved rod domain of IF proteins (Geisler et aL, 1985; Napolitano et a/.,

1987; Lees et aL, 1988), the long C-terminal tail extensions of NF-M and NF-H

extend as sidearms from native or reconstituted IFs (MuHigan et aL, 1991).

Based upon periodic decoration of vimentin IFs from avian erythrocytes

(Granger and Lazarides, 1982) or lens cells (Granger and Lazarides, 1984) with synemin antibodies, which indicates that at least some of the synemin molecule is located at the periphery of these IFs, analogies have been drawn between synemin and the larger NF peptides, especially NF-H. Although NF-L efficiently 108 assembles Into IPs, efficiency of the larger two, NF-M and NF-H, alone or in

combinations, to assemble into IFs decreases with their increasing size (e.g.,

NF-H fails by itself to form typical IFs) (Hirokawa et a!., 1984; Balin and Lee,

1991). We tested the ability of synemin to form filaments under conditions in

which NF-H can be induced to self-assemble in vitro into very short, ~ S-shapéd

or wavy, kinked filaments of --7-10 nm diameter (Gardner et a!., 1984;

Troncoso et al., 1988; Balin and Lee, 1991). Interestingly, similar structures

were formed in our study of synemin (Fig. 3D). Thus, the suggestion that

synemin may co-assemble or attach peripherally to desmin IFs and serve to

connect IFs or IFs to other cellular structures or organelles (Granger and

Lazarides, 1982, 1984) seems plausible. A more precise relationship of synemin to NF-H, however, is tenuous because synemin does not appear to contain the conserved rod domain common to IF proteins (Pruss et a!., 1981), and NF-H exists as a monomeric molecule in low ionic strength, slightly alkaline solutions (Cohlberg et at., 1987), whereas our results show that synemin is a dimeric molecule.

Identification of Synemin in Mammalian Muscles—Synemin was originally identified in avian smooth muscle and in developing and mature avian skeletal muscle by antibody localization and immunoautoradiography (Granger and

Lazarides, 1980). Sandoval et aL, (1983) subsequently reported the presence of a reactive synemin band at ~230 kDa in fetal and adult chicken cardiac, smooth, and skeletal muscles by immunoautoradiography. Antibody localization 109 studies from the same laboratory (Price and Lazarides, 1983), however,

demonstrated that synemin is present in all avian developing muscle cells, but

only in adult avian skeletal and smooth muscle cells. In adult cardiac muscle,

synemin was absent and replaced by paranemin. The differences in results in adult cardiac muscle from the same laboratory were not addressed. We have unequivocally identified synemin in all three adult avian muscle samples, including cardiac muscle, both by Western blot analysis and by antibody localization on myofibrils and/or cryostat sections.

Granger and Lazarides (1980) also have reported that synemin is absent in adult mamrpalian striated and smooth muscle cells and in mammalian lens cells

(Granger and Lazarides, 1984) and, thus, a mammalian homolog of synemin has not previously been found. We have identified a mammalian synemin homolog by using polyclonal anti-synemin antibodies raised against highly-purified avian gizzard synemin. These antibodies labeled two bands at ~225 kDa and ~195 kDa in adult porcine striated (skeletal and cardiac) and smooth muscle samples by Western blot analysis. We have not determined the nature of the relationship between the two proteins (225 kDa and 195 kDa). The ~195 kDa protein may be a proteolytic product derived from the ~225 kDa protein, or may represent a different size isoform. A prominent band at ~ 190-200 kDa has been described as a major proteolytic product of avian synemin (Sandoval et al., 1983), and by analogy, this lower band seen in blots of our mammalian muscle samples may be a similar breakdown product. To substantiate that the 110 ~225 kDa and ~ 195 kDa proteins from mammalian muscle are synemin

homologs, a cytoskeletal-enriched acetone powder was prepared from porcine

stomach muscle in a manner identical to that described in this report for avian

gizzard muscle. Extraction of porcine smooth muscle acetone powder with

buffer B and subsequent hydroxyapatite (only one column was used) and DEAE-

Sephacel chromatography resulted in fractions containing the reactive - 225

kDa and ~ 195 kDa proteins that eluted where gizzard synemin routinely eluted.

The presence of synemin (or a homolog) in mammalian muscle cells also was demonstrated by immunofluorescence labeling of isolated muscle myofibrils and of cryosections of muscle. The anti-synemin antibodies specifically label the myofibrillar Z-lines of both adult avian and porcine striated (skeletal and cardiac) muscle samples in a punctate pattern. This Z-line labeling pattern of synemin is in agreement with the distribution and the immunofluorescence labeling pattern shown for synemin by Granger and Lazarides (1980) for avian skeletal muscle myofibrils. Those investigators demonstrated that synemin exists at the periphery of the Z-disk, forming a network of interlinking rings at the Z-plane, identical to the distribution of desmin IPs (Granger and Lazarides, 1980).

Localization of the cryosections was included in our study to rule out adventitious binding of any non-muscle cell reactive antigens that conceivably could occur during myofibril isolation procedures.

We have noted in our study that although anti-avian synemin antibodies label all three adult mammalian and avian muscle types, including cardiac, the Ill intensity of immunofluorescence labeling in both species was somewhat weaker

with the cardiac muscle samples than that observed with the skeletal muscle

samples.

We have noted that two proteins of ~220 kDa and ~200 kDa, named

skelemins (Price, 1984), have been isolated from a cytoskeletal fraction from bovine myocardium and shown also to be present in bovine skeletal and smooth muscle in a 1:1 molar ratio (Price, 1987). Studies conducted by Price (1987), however, have shown that skelemins are different from synemin by immunological, two-dimensional peptide mapping, pi, molecular weight, and tissue distribution criteria. Antibodies raised against the skelemins specifically label mammalian myofibrillar M-lines and do not label reactive proteins in chicken muscle (Price, 1984, 1987). Based on those results, and the specific

Z-line labeling of the anti-avian-synemin antibodies used in this study, it is evident that the ~225 kDa and -195 kDa proteins identified in this study are distinct from the skelemins.

The role of synemin is unknown, although it has been proposed that synemin may regulate the extent of crosslinking of IPs into loose networks and/or mediate interactions of IPs with other intracellular organelles or with the plasma membrane (Granger and Lazarides, 1982, 1984). We have shown in this study that synemin colocalizes with desmin at the myofibrillar Z-lines of both adult mammalian and avian striated muscle. It has not been clear as to whether desmin IPs are in direct contact with the myofibrillar Z-lines. Some have 112 suggested that there may be small gaps between the edge of the Z-lines and IPs

(Tokuyasu et aL, 1985; Bard and Pranzini-Armstrong, 1991). M.M. Bllak et at.

(1991b) have recently shown that IPs come to within at least 8 nm of the Z-line edges, a distance that may easily be spanned by a crosslinking protein. Our results suggest that synemin is at least present in the location necessary for it to act as an IP/myofibrillar crosslinker, and we have recently found that synemin can interact in vitro with both desmin and a-actinin (S.R. Bilak et aL, 1991). 113 ACKNOWLEDGMENTS

Monoclonal anti-desmin (D3) was purchased from the Developmental Studies

Hybridoma Bank (contract no. N01-HD-6-2915). We thank Marlene Bright,

Masako Bilak, Mary Sue Mayes, and Brad Gerndt for technical assistance. This work was supported, in part, by grants from the Muscular Dystrophy

Association, USDA Competitive Grants Program (Award 89-37265-4441),

National Live Stock and Meat Board, and the American Heart Association, Iowa

Affiliate. This is Journal Paper J-14737 of the Iowa Agriculture and Home

Economics Experiment Station, Ames, lA 50011, projects 2921, 2930, and

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SECTION II. INTERACTIONS OF PURIFIED SYNEMIN

WITH MUSCLE Z-LINE PROTEINS 122

INTERACTIONS OF PURIFIED SYNEMIN

WITH MUSCLE Z-LINE PROTEINS

Stephan R. Bilak, Richard M. Robson, Masako M. Bilak, and Marvin H. Stromer

Muscle Biology Group, Departments of Animal Science

and of Biochemistry and Biophysics

Iowa State University

Ames, Iowa 50011, U.S.A

Contact Individual: Dr. Richard M. Robson

Muscle Biology Group, Iowa State University

Ames, Iowa 50011

Telephone: (515) 294-5036

FAX: (515) 294-8181

Running Title: Interactions of Synemin with Z-line Proteins 123 ABSTRACT

In vertebrate striated muscle cells, desmin-containing intermediate filaments

(IPs) primarily run transversely to the long axis of the cell at the myofibrillar Z-

iines and possibly link adjacent myofibrils to each other. The precise nature of

the association between the IPs and the myofibrillar Z-lines has not been

elucidated. We have examined the interactions of synemin, an IF-associated

protein (IFAP) that is colocaiized with desmin at the edge of Z-lines, with

desmin, the major IF protein in adult striated muscle cells, and with a-actinin, a

major protein component of the myofibrillar Z-line. When purified desmin and

purified synemin are combined and dialyzed against IF-assembly buffer, the ability of desmin to assemble into long IFs is significantly altered [e.g., at a ratio of 5:1 {desmin:synemin, w/w), the filaments are shortened, with many of them measuring ~0.39 //m and -0.17 //m in length]. As the amount of synemin relative to desmin increases, the filaments become progressively shorter (the shortest measuring ~ 50-70 nm), and many of these very short filaments display an unraveled or frayed appearance. Immunogoid labeling of desmin/synemin mixtures with polyclonal anti-synemin antibodies shows that synemin is present both along the desmin IFs and at points of filament intersection. Solid-phase binding assays, using both dot blot and Western blot analyses, show that synemin can bind to both desmin and or-actinin. These results indicate that synemin affects IF assembly dynamics, and give support for the suggestion that synemin may help link IFs to myofibrillar Z-lines. 124

Key words; synemin, desmin, intermediate filaments, muscle Z-lines, cytoskeleton, myofibrils 125

INTRODUCTION

The intermediate-sized filaments (IPs) of ~10 nm diameter [Ishikawa et al.,

1968] are one of the three major cytoskeletal classes of filaments found in nearly all vertebrate cells [Lazarides, 1982; Steinert and Roop, 1988]. Desmin, a sequence type III IF protein [Robson, 1989], is the major constituent of the

IPs present in mature skeletal, cardiac, visceral smooth and many vascular smooth muscle cells [Lazarides and Hubbard, 1976; Schollmeyer et al., 1976;

Stromer, 1990], and desmin has been purified from visceral smooth, skeletal and cardiac muscles [Huiatt et al., 1980; O'Shea et al., 1981; Hartzer, 1984].

Although much progress has been made concerning the individual properties of IF proteins [Stewart, 1990], little is yet known with certainty regarding their specific cellular function [Bloemendal and Pieper, 1989; Klymkowsky et al.,

1989; Skalli and Goldman, 1991]. This is also the case for desmin, especially with regard to developing muscle cells [Schultheiss et al., 1991; Tao and Ip,

1991]. The major role suggested for desmin IPs in adult striated muscle cells is cytoskeletal in nature, i.e., to maintain lateral registry and integrity of the myofibrils by providing a framework which links adjacent myofibrils together

[Lazarides, 1980; Richardson et al., 1981].

Immunofluorescence [Lazarides and Hubbard, 1976; Lazarides and Granger,

1978] and immunoelectron [Richardson et al., 1981; Tokuyasu et al., 1983a,b;

M.M. Bilak et al., 1991a] microscope studies have demonstrated that, in mature striated muscle cells, desmin IPs are primarily transversely oriented to the long 126 myofibrillar axis and located at the periphery of the Z-lines. However, the exact

relationship of IPs to the myofibrillar Z-lines is not clear, and some investigators

[Tokuyasu et al., 1985; Bard and Franzini-Armstrong, 1991] have suggested

that there may not be direct attachment of IPs to the edge of the Z-line. Recent

studies from our laboratory, including examination of routine cross-sections and

of stereo pairs of transmission electron micrographs, indicate that IPs surround

the myofibrils at their Z-lines, rather than penetrate into the Z-line domain.

Although the IPs may be directly attached at some positions to the Z-lines,

there is sometimes an apparent gap of ~ 8 nm or more between IPs and the

edges of the myofibrillar Z-line [M.M. Bilak et al., 1991b]. Thus, it has been

suggested that additional crosslinking components may be involved in the

interaction between the IPs and the Z-lines [Tokuyasu et al., 1985; M.M. Bilak

et al., 1991a,b]. One possible candidate for such a crosslinking function is

synemin, a 230 kD IP-associated protein (IPAP). Synemin, was originally

identified in avian developing and mature muscle cells [Granger and Lazarides,

1980]. Synemin has been shown by immunofluorescence [Granger and

Lazarides, 1980; S.R. Bilak et al., 1991] and immunoelectron [M.M. Bilak et al.,

1991a] microscope studies to be colocalized with desmin at the periphery of

mature avian and mammalian striated muscle Z-lines. As part of our continuing

interest in understanding the Z-line structure [Yamaguchi et al., 1985; Goll et al., 1991], and to provide evidence to support the suggestion that IPs interact

with myofibrillar Z-lines, we have examined the ability of synemin to interact 127 with both desmin and a-actinin. The ability of synemin to interact with desmin was assayed by examining synemin's effect on desmin assembly by electron microscopy. We also present biochemical (immunoblot) evidence that synemin can bind to both desmin and o-actinin, a major Integral Z-line protein. A preliminary report, Including part of this study, was presented at the 1990

American Society for Cell Biology meeting [Bilak et al., 1990]. 128

MATERIALS AND METHODS

Protein Preparations

Desmin and synemin were purified from fresh turkey gizzard smooth muscle

according to the procedures of Huiatt et al. [1980] and S.R. Bilak et al. [1991],

respectively. a-Actinin was prepared from turkey gizzard smooth muscle

according to the procedures of Robson et al. [1970] and Suzuki et al. [1976].

The 55 kD fragment of a-actinin was prepared by treating purified gizzard a-

actinin with trypsin (Sigma Chemical Company, St. Louis, MO; Type IV) with a

1:25 (w/w) enzyme-to-o-actinin ratio in 20 mM Tris-acetate, pH 7.5, at 25°C.

After 90 min, the digestion was terminated by addition of soybean trypsin

inhibitor (Sigma) (1:1 =trypsin:trypsin inhibitor, w/w), and the digestion

products were separated by TSK-Gel DEAE-Toyopearl (Toyo Soda

Manufacturing Co., Ltd., Tokyo, Japan) chromatography. The column (0.9 cm

X 29 cm) was poured and equilibrated in 20 mM Tris-acetate, pH 7.5. After

loading, the column was eluted with a linear gradient made from 60 ml each of

20 mM Tris-acetate, pH 7.5, and 20 mM Tris-acetate, pH 7.5, that contained

500 mM KCI. Fractions containing only the 55 kD product, as determined by

SDS-PAGE, were pooled and used herein. This a-actinin fragment consists of

the central, rod-like, anti-parallel domain (two subunits of ~55 kD each) and contains four spectrin-like repeats [Arakawa et al., 1985; Blanchard et al.,

1989; Davison et al., 1989].

Protein concentrations were determined by the modified Lowry method 129 [Lowry et al., 1951], by using a procedure supplied by Sigma Chemical

Company (procedure no. P 5656).

Filament Formation Studies

Immediately before use, highly purified samples of desmin and synemin were

individually dialyzed extensively against 0.1% (v/v) /9-mercaptoethanol (MCE),

10 mM Tris-HCI, pH 8.5, and then against 10 mM Tris-HCI, pH 8.5, at 4°C and

clarified (183,000 x g^ax for 1 hr). Samples were prepared by mixing desmin

and synemin at specific ratios (w/w) in 10 mM Tris-HCI, pH 8.5, with desmin

kept at a constant protein concentration of 0.1 mg/ml. The protein mixtures

were dialyzed for 16-18 hr at 4°C against an "IF-assembly buffer" (100 mM

NaCI, 1 mM MgClj, 10 mM imidazole-HCI, pH 7.0). The samples were then examined by negative staining electron microscopy as follows: (1) a drop of protein suspension was placed on a glow-discharged carbon-coated (400-mesh) grid for 1 min, gently washed with 30 drops of glass-distilled water, and the excess water was removed by touching the grid with the edge of filter paper, and (2) the grid was stained with 2% aqueous uranyl acetate for 1 min, air- dried after removal of excess stain, and examined in a JEOL JEM-100CXII transmission electron microscope (TEM) operated at 80 kV. A template of ruled lines was randomly oriented over electron micrographs, and diameters and lengths of filaments that fit into every third square were measured with a measuring magnifier. 130 Immunogold Labeling of Synemin/Desmin Assembly Products

Desmin assembly, done as described above, in the presence of differing

amounts of synemin, was examined by a protein A-gold labeling procedure as follows: (1) samples of defined protein mixtures were placed on a 400-mesh carbori-coated grid and, after 1 min, the grid was gently rinsed with 10 drops of glass-distilled HjO and then floated, face down, on a droplet of cytochrome-c

(aqueous) (Sigma) (5 //g/ml) (on a sheet of parafilm) for 2 min, (2) the grid was rinsed with 10 drops of phosphate-buffered saline (PBS) and floated on a droplet of affinity-purified anti-synemin polyclonal antibodies (diluted 1:10 with

PBS) for 10 min at 24°C, (3) the grid was washed by floating on 10 sequential droplets of PBS (10 s each) and then incubated for 5 min with protein-A gold complex (5-nm complex from Amersham, UK, or 10-nm complex from Sigma) which had been diluted 1:20 with 1 % Teleostean gelatin (Sigma) in PBS, at

24°C, and (5) the grid was washed with PBS as in step (3), stained with 2% aqueous uranyl acetate, and examined in a JEM as above.

Solid-Phase Binding Assays

For the dot blot analysis, 25-30 //g of synemin, desmin, or bovine serum albumin (BSA) control, all present in 10 mM Tris-HCI, pH 8.5 (overlay buffer for these proteins), or a-actinin or BSA control, present in 100 mM KCI, 20 mM sodium bicarbonate, pH 7.5 (overlay buffer for these proteins), were spotted directly onto nitrocellulose (NC) (Bio-Rad Laboratories, Richmond, CA) by using 131 a dot blot manifold (Bio-Rad). For Western blot (gel blotting) analysis, samples

of whole turkey gizzard smooth muscle homogenate, purified synemin, desmin, a-actinin, or 55 kD a-actinin fragment were subjected to SDS-PAGE according to Laemmli [1970], except that mini-gels were run using the Mighty Small II SE

250 electrophoresis cell (Hoefer Scientific Instrument, San Francisco, CA) at 30 mA. Proteins were electrophoretically transferred to NC membranes (Bio-Rad) by using the Bio-Rad transblot apparatus for 1 hr at 90 V according to Towbin et al. [1979]. Reversible staining with Ponceau S (1%, w/v in 1% acetic acid)

(Sigma) was used to ensure that proteins were properly spotted in the dot blot assay, and to ensure protein transfer in the Western blot assay. The following steps, used in common for both dot blot and Western blot analyses, were as follows: (1) the blots were blocked with 2% (w/v) Blotto (non-fat, dry milk), diluted in overlay buffer, for 45 min and then rinsed in appropriate overlay buffer (no Blotto); (2) the blots were overlayed with either desmin or synemin

(present in 10 mM Tris-HCI, pH 8.5), or alpha-actinin (present in 100 mM KCI,

20 mM sodium bicarbonate, pH 7.5) (all proteins in the overlay buffer were at

0.1 mg/ml); (3) the overlays were done at 4°C with constant agitation for 24 hr; (4) the blots were washed for 30 min with appropriate overlay buffer, and then for 45 min with Tris-buffered saline (TBS) (500 mM NaCI, 20 mM Tris-HCI, pH 7.0) containing 1 % (w/v) Blotto; (5) the blots were incubated for 4 hr at

24°C with a primary antibody directed against the protein used in the overlay

(see below); and (7) after washing with 1% (w/v) Blotto in TBS (3x15 min), 132 the blots were incubated with peroxidase-conjugated anti-rabbit IgG (for anti-

synemin and anti-desmin antibodies) or anti-mouse IgM (for anti-a-actinin

antibodies), diluted 1:1,000 in TBS containing 1% (w/v) Blotto, washed 3x15

min, and developed with 4-chloro-1-naphthol and HjOj reagents (Sigma).

Immunological Reagents

Rabbit anti-desmin polyclonal antibodies to highly purified porcine skeletal

muscle desmin [O'Shea et al., 1981] were prepared by the procedure of

Richardson et al. [1981]. Rabbit anti-synemin polyclonal antibodies to highly purified turkey gizzard synemin were prepared as described by S R. Bilak et al.

[1991]. The polyclonal anti-synemin antibodies used herein were affinity purified by DEAE-Affi-Gel Blue (Bio-Rad) chromatography. Monoclonal anti-a- actinin antibodies were purchased from Sigma Chemical Company. The polyclonal anti-synemin and polyclonal anti-desmin antibodies used have previously been shown to specifically label at the myofibrillar Z-lines of mature chicken and porcine skeletal and cardiac myofibrils by indirect immunofluorescence [S.R. Bilak et al., 1991] and immunoelectron [M.M. Bilak et al. 1991a] microscopy. The monospecificity of all antibodies used in this study also was confirmed herein by Western blot analysis by using whole turkey gizzard smooth muscle homogenate, and synemin, desmin, and a-actinin purified from turkey gizzard smooth muscle (Fig. 1). The anti-desmin polyclonal antibodies were diluted 1:250, and the polyclonal anti-synemin and monoclonal 133 anti-a-actinin antibodies were diluted 1:500, all with 1% (w/v) Blotto in TBS.

After washing 3x15 min as above, the blots were incubated with appropriate

peroxidase-conjugated antibody and developed as above.

The anti-a-actinin antibodies recognized purified avian smooth muscle a- actinin (Fig. IB, lane 4) and only the a-actinin present in the smooth muscle homogenate (Fig. IB, lane 1). The anti-desmin antibodies recognized purified avian smooth muscle desmin (Fig. 1C, lane 3) and only the desmin present in the smooth muscle homogenate (Fig. 1C, lane 1). The polyclonal anti-synemin antibodies recognized purified avian smooth muscle synemin (Fig. ID, lane 2) and only the synemin present in the smooth muscle homogenate (Fig. 10, lane

1). Fig. 1. Characterization of antibodies used in this study by Western blot analysis. (A) SDS-polyacrylamide gel stained with Coomassie Blue; (B) Western blot of a duplicate gel shown in (A) incubated with anti-a-actinin; (C) Western blot of a duplicate gel shown in (A) incubated with anti-desmin; (D) Western blot of a duplicate gel shown in (A) incubated with anti-synemin. Lane 1 = turkey gizzard homogenate; lane 2 = purified turkey gizzard synemin (S.R. Bilak et al., 1991); lane 3 = purified turkey gizzard desmin [Huiattet al., 1980]; and, lane 4 = purified turkey gizzard a-actinin [Robson et al., 1970]. As shown in (B), anti-a-actinin antibodies recognize a-actinin present in gizzard homogenate (lane 1) as well as purified a-actinin (lane 4), but do not crossreact with either desmin (lanes 1,3) or synemin (lanes 1,2). As shown in (C), anti- desmin antibodies recognize desmin present in gizzard homogenate (lane 1 ) as well as purified desmin (lane 3), but not synemin (lanes 1,2) or a-actinin (lanes 1,4). As shown in (D), anti-synemin antibodies recognize synemin present in gizzard homogenate (lane 1) as well as purified synemin (lane 2), but not desmin (lanes, 1,3) or a-actinin (lanes 1,4). 123 4

230^ 200^

100—*^ 100—

k •" 136 RESULTS

Studies showing colocalization of synemin witli type III IF proteins [Granger

and Lazarides, 1980, 1982, 1984; Granger et al., 1982; M.M. BIlak et al.,

1991a; S.R. Bilak et al., 1991] suggest that synemin and desmin may interact,

but evidence for a direct interaction has been difficult to obtain [Sandoval et al.,

1983]. Thus, we have examined the ability of purified synemin to interact with

purified desmin in vitro. The assembly of desmin IPs was used as one very

sensitive indicator of synemin/desmin interaction. As shown in Figure 2A

(inset), purified desmin self-assembles into IFs (approximately 10 nm in

diameter) that are long, smooth-sided, and uniform in morphology. In general,

the filaments are highly intertwined, but some filaments can be followed for

over 1 //m. The results obtained with the identical assembly conditions, but in

the presence of purified synemin, are shown in Figure 2A-C. Figure 2A shows

a 5:1 desmin:synemin (w/w) mixture after dialysis against the IF-assembly

buffer. Although the filaments are ~ 10 nm in diameter, they are significantly

shorter than the control filaments (inset). Measurements of the filament lengths

revealed that these shorter filaments were not random, but instead were

present in size (length) categories of ~0.39 ±0.007 //m (mean ±standard error

of the mean; n = 22) (double arrows), -0.17±0.002 /ym (n =98) (triple arrowheads), and -0.11 ±0.001 //m (n = 194) (double arrowheads). Small IF assembly intermediates, protofibrils or protofilaments [Stromer et al., 1981; Ip et al., 1985b], were nearly absent. As the amount of synemin is increased, the Fig. 2. Electron micrographs of negatively stained IFs assembled from desmin in the absence and presence of synemin. A (inset), Filaments formed from purified desmin (0.1 mg/ml) after dialysis against IF-assembly buffer. The filaments are long, uniform in morphology and have a diameter of -10 nm. A- C, Filaments formed from purified desmin (0.1 mg/ml) and purified synemin at selected ratios (w/w) after dialysis against IF-assembly buffer. A, Synemin:desmin = 1:5 (w/w) ratio. Overall, the filaments are -10 nm in diameter, but shorter. Filament lengths vary [e.g., -0.39 //m (double arrows), -0.17 //m (triple arrowheads), and -0.11 //m (double arrowheads) in length]. Structures smaller than short, full width IFs (e.g., protofibrils/protofilaments) are virtually absent at this ratio. B, synemin;desmin = 1:1 (w/w) ratio. Only short filaments are present [-0.17 ;ym (triple arrowheads), -0.11 //m (double arrowheads), and - 50-70 nm (arrowheads) in length]. A few of the filaments are -10 nm in diameter, but many of the filaments appear to be slightly unravelled (asterisks) and have diameters larger than -10 nm. Some smaller assembly intermediates [protofibrils (arrows)/protofilaments (small arrows)] are present. C, Synemin:desmin = 2:1 (w/w) ratio. Overall, the filaments are very short, with many measuring -0.11 //m (double arrowheads) and -50-70 nm (arrowheads) in length. The filament diameters are not uniform. A large number of smaller IF assembly intermediates [protofibrils(arrows)/protofilaments (small arrows)] also are present. Bar in C is for A-C=0.1 yt/m. m 139 number of the shorter classes of filaments progressively increases (Fig. 2B,C).

At a desmin:synemin ratio of 1:1 (w/w), very few filaments longer than ~0.17

/jm (triple arrowheads) are present, and the majority of the filaments measure

-0.11 yt/m (double arrowheads) and ~50-70 nm (arrowheads) in length. In

most cases, these short filaments are slightly unraveled and have diameters

larger that 10 nm (asterisks). In addition, there are many smaller IF assembly

intermediates [protofibrils (arrows) and protofilaments (small arrows)] present.

The assembly products resulting from addition of 1 part desmin to 2 parts

synemin (w/w) and dialysis against IF-assembly buffer are shown in Figure 2C.

At this ratio of desmin to synemin, long IFs (5:0.39 fjm) are absent. Most of

the filaments measure -0.11 fjm (double arrowheads) and ~ 50-70 nm

(arrowheads) in length, and a large number of protofibrils (arrows) and protofilaments (small arrows) are present. We have shown elsewhere that under these buffer conditions, in which desmin forms long IFs [Huiatt et al.,

1980; Fig. 2A (inset)], synemin does not [S.R. Bilak et al., 1991]. Instead, synemin forms irregular aggregates (-15-25 nm in diameter) composed of several -11 nm diameter spheres. These particles are essentially absent in the results shown in Figure 2B,C.

IFs assembled from desmin in the presence of different amounts of synemin, and then sequentially incubated with pre-immune serum or anti-synemin, and protein A-gold complex, are shown in Figure 3. Control samples, treated on a grid with pre-immune serum followed by incubation with protein A-gold, all Fig. 3. Electron micrographs of negatively stained IPs assembled from desmin in the presence of synemin and sequentially incubated with anti-synemin and protein A-gold complex. A-C, Filaments formed from purified desmin (0.1 mg/ml) and purified synemin at a 10:1 (w/w) ratio. The IFs are generally long and fairly uniform in diameter. A, Control incubated with pre-immune serum and protein A-gold (10 nm), demonstrating very low non-specific background labeling (arrow). B and C, IFs incubated with anti-synemin and protein A-gold (10 nm). Labeling was evident at points of filament intersection (B and C, arrows), and along the filaments (0, arrowhead). D-l, Filaments formed from purified desmin (0.1 mg/ml) and purified synemin at a 5:1 (w/w) ratio. The IFs are generally shorter, but still fairly uniform in diameter. D, Control incubated with pre-immune serum and protein A-gold (10 nm), demonstrating virtually no background labeling. E-l, IFs incubated with anti-synemin and protein A-gold (10 nm). Labeling was evident along the short IFs (E, F, and I, arrows) and often ~0.1 //m from the ends of filaments (E-H, arrowheads). Bar shown in I is for micrographs E-l, and equals 0.125 //m. J-L, Filaments formed from purified desmin (0.1 mg/ml) and synemin at a 1:1 (w/w) ratio. The IFs are generally very short and not uniform in diameter. J, Control incubated with pre-immune serum and protein A-gold (5 nm), demonstrating very low background labeling (arrow). K and L, IFs incubated with anti-synemin and protein A-gold (5 nm). Virtually all of the short filaments (arrows) were labeled, and coated with antibodies as indicated by their fuzzy appearance. Bar shown in L is for all micrographs except E-l, and equals 0.25 /ym. w

•s? 142 demonstrate very low non-specific labeling (arrow) as shown in Figure 3A,D,J.

A 10:1 desmin:synemin (w/w) mixture, dialyzed against IF-assembly buffer, yields fairly uniform diameter, non-truncated filaments (Fig. 3A-C). Labeling of these synemin/desmin mixtures with anti-synemin and protein A-gold suggests that synemin is associated with the filaments, primarily at points of filament intersection (Fig. 3B,C; arrows) and along the filaments (Fig. 3C; arrowhead).

Results of 5:1 (w/w; desmin:synemin) mixtures treated on grids with anti- synemin and protein A-gold are shown in Figure 3E-I. Labeling is common along the IFs (Fig. 3E,F,I; arrows), often ~70 nm from the ends of filaments

(Fig. 3E-H; arrowheads). Figure 3K,L shows examples of 1:1 mixtures (w/w) of desmin and synemin, incubated with anti-synemin and protein A-gold. At this ratio, the filaments are very short, and labeling is very extensive. Virtually all of the short filamentous structures exhibit a fuzzy appearance, presumably due to binding of the anti-synemin antibodies.

The results on desmin assembly in the presence of synemin shown in Figures

2 and 3 indicate that there is a desmin/synemin interaction. To provide biochemical evidence for this interaction, we used two types of solid-phase binding assays. In these assays, a-actinin, a major integral Z-line protein, also was included. The results of dot blot assays are shown in Figure 4. In Panel A,

NC strips containing spots of desmin, synemin, and BSA were overlayed with synemin, and then incubated with polyclonal anti-synemin antibodies followed by incubation with goat-anti-rabbit IgG-horseradish peroxidase. Control NC Fig. 4. Dot immunoblot overlay assay demonstrating interactions among synemin, desmin, and a-actinin. 25-30 //g of purified desmin, synemin, and control bovine serum albumin (BSA) were spotted directly onto nitrocellulose (NC) strips and overlaid with either synemin (panel A) or desmin (panel C) in 10 mM Tris-HCI, pH 8.5, or with a-actinin (panel E) in 100 mM KCI, 20 mM NaHCOg, pH 7.5. The NC strips were sequentially washed, incubated with primary antibodies specific for the protein used in the overlay, and incubated with appropriate secondary antibodies linked to horseradish peroxidase as described in the Materials and Methods. In all panels (A-F), two representative NC strips are shown. Panel A, NC strips overlayed with synemin. Panel B, Control (not overlaid with synemin) for NC strips shown in (A). Anti-synemin labels the synemin wells (A and B) and synemin bound to the desmin wells (A), which shows that synemin binds to desmin. Anti-synemin does not label the desmin wells in (B) or the BSA wells (A or B). Panel 0, NC strips overlayed with desmin. Panel D, Control (not overlaid with desmin) for NC strips shown in (0). Anti-desmin labels the desmin wells (0 and D) and desmin bound to the synemin wells (0), which shows that desmin binds to synemin. Anti-desmin does not label the synemin wells in (D) or the BSA wells (0 or D). Panel E, NC strips overlayed with a-actinin. Panel F, Control (not overlaid with a-actinin; only a-actinin, desmin, and synemin were spotted on the NC strips). Anti-a- actinin labels the a-actinin control wells (F) and a-actinin bound to the desmin and synemin wells (E), which indicates that a-actinin binds to both desmin and synemin. Anti-a-actinin does not bind to the BSA wells in (E) or the desmin or synemin wells in (F). A B desmin-* desmin—» '•s i f \ ; 'h . 0# synemin-* synemin—

BSA— BSA--

uJù t desmin desmin—» synemin synemin—»

BSA-»

desmin—» a-ochnin—» synemin—» desmin—»

synemin—» 145 strips for those shown in Panel A are shown in Figure 4, Panel B. The controls

were not overlayed with synemin, but were incubated with anti-synemin

antibodies and the secondary antibody-conjugate. Anti-synemin labels the

synemin wells (Panels A and B; labeling of synemin in Panel B serves as an

internal positive control) and synemin bound to the desmin wells (Panel A),

which indicates that synemin binds to desmin. Anti-synemin does not label the

desmin wells in Panel B or the BSA wells (Panels A or B). In Figure 4, Panel C,

NC strips containing desmin, synemin, and BSA spots were overlayed with

desmin and incubated with polyclonal anti-desmin antibodies followed by incubation with goat-anti-rabbit IgG-horseradish peroxidase. The control NC strips (not overlayed with desmin, but incubated with anti-desmin antibodies and the secondary antibody-conjugate) for those shown in Panel C, are shown in Figure 4, Panel D. Anti-desmin labels the desmin wells (Panels C and D; labeling of desmin in Panel D serves as an internal positive control) and desmin bound to the synemin wells (Panel C), which shows that desmin binds to synemin. Anti-desmin does not label the synemin wells in Panel D or the BSA wells (Panels C and D). Since or-actinin is an important integral protein of the muscle Z-line (Goll et al., 1991), we were interested in examining its ability to interact with synemin and/or desmin. In Figure 4, Panel E, NC strips containing desmin, synemin, and BSA spots were overlayed with a-actinin and incubated with monoclonal anti-a-actinin antibodies followed by incubation with goat-anti- mouse IgM-horseradish peroxidase. The control NC strips (not overlayed with 146 a-actinin, but incubated with anti-a-actinin antibodies and the secondary

antibody-conjugate), are shown in Figure 4, Panel F. Anti-a-actinin labels the a-

actinin positive control wells (Panel F) and both the desmin and synemin wells

in Panel E, which indicates that a-actinin binds to both desmin and synemin.

Anti-a-actinin does not bind to the BSA wells in Panel E (the small amount of

reaction product shown was within normal background limits) or the desmin or the synemin wells in Panel F. Results of solid-phase binding assays, in

which samples of whole turkey gizzard smooth muscle homogenate, synemin, desmin, and a-actinin were electroblotted from polyacrylamide gels onto NC, overlayed with desmin, synemin, or a-actinin, then incubated with appropriate secondary antibody-conjugate and developed, are shown in Figure 5. Figure 5A shows a Coomassie Blue stained SDS-polyacrylamide gel (10%) that contains the smooth muscle homogenate lane (lane G), purified gizzard synemin (lane 1 ), purified gizzard desmin (lane 2), purified gizzard a-actinin (lane 3), and a purified

55 kD proteolytic fragment of gizzard a-actinin (lane 4). The smooth muscle homogenate (lane G) was useful in that it served as molecular weight standards and as both negative and positive controls, because it contained major myofibrillar/cytoskeletal proteins such as filamin, myosin heavy chain, desmin, and actin. A Western blot of a duplicate gel to that shown in Figure 5A, overlayed with synemin and incubated with polyclonal anti-synemin antibodies and then with goat-anti-rabbit IgG-horseradish peroxidase, is shown in Figure

58. As expected, the antibodies label synemin in the smooth muscle Fig. 5. Western blot overlay assay demonstrating interactions among synemin, desmin, and ûr-actinin. Samples were run on SOS-polyacrylamide gels (10%) (A), electrophoretically transferred to nitrocellulose membranes and overlayed

with either synemin (B) or desmin (C) in 10 mM Tris-HCI, pH 8.5, or with a- actinin (D) in 100 mM KCI, 20 mM NaCHOg, pH 7.5. The blots (B-D) were sequentially washed, incubated with primary antibodies specific for the protein used in the overlay, incubated with appropriate secondary antibodies linked to horseradish peroxidase, and developed. A, SDS-polyacrylamide gel stained with Coomassie Blue: lane G = whole turkey gizzard homogenate showing filamin (250 kD), myosin (205 kD), a-actinin (100 kD), desmin (53 kD), and actin (42 kD); lane 1 = purified synemin (230 kD); lane 2 = purified desmin; lane 3 = purified a-actinin; lane 4 = isolated 55 kD fragment of a-actinin. B, Western blot of a duplicate gel to that shown in (A) overlayed with synemin and incubated with anti-synemin. The antibodies label synemin present in the whole turkey gizzard homogenate (lane G) and the purified synemin control (lane 1). The antibodies also label synemin that has bound to: desmin in the whole turkey gizzard homogenate (lane G), purified desmin (lane 2), purified a-actinin (lane 3), and 55 kD a-actinin fragment (lane 4). C, Western blot of a duplicate gel to that shown in (A) overlayed with desmin and incubated with anti-desmin. The antibodies label desmin in the whole turkey gizzard homogenate (lane G) and the purified desmin control (lane 2). The antibodies also label desmin that has bound to: synemin in the whole turkey gizzard homogenate (lane G), purified synemin (lane 1), purified a-actinin (lane 3), and the 55 kD a-actinin fragment (lane 4). D, Western blot of a duplicate gel (minus the 55 kD a-actinin fragrnent lane) to that shown in (A), overlaid with a-actinin and incubated with anti-a-actinin. The antibodies label a-actinin present in the whole turkey gizzard homogenate (lane G) and the purified a-actinin control (lane 3). The antibodies also label a-actinin that has bound to: synemin and desmin bands in the whole turkey gizzard homogenate (lane G), purified synemin (lane 1) and purified desmin (lane 2). 230-:^ 230-^

« 100-^ lOOH

ffe's®

53-^

G 1 2 3

230^ 230—

100"* 100-*^ 149 homogenate (lane G) as well as the synemin control (lane 1). The antl-synemin

also labels synemin that has bound to: (a) desmin present in the smooth muscle

homogenate (lane G), (b) purified desmin (lane 2), (c) purified a-actinin (lane 3),

and (d) the 55 kD fragment of a-actinin (lane 4). Synemin binding to the small

amount of a-actinin in lane G is below the detection limit, but binding to this

band was obtained if the gel was overloaded (results not shown). A Western

blot of a duplicate gel to that shown in Figure 5A, overlayed with desmin and

incubated with polyclonal anti-desmin antibodies and then with goat-anti-rabbit

IgG-horseradish peroxidase, is shown in Figure 5C. As expected, the antibodies

label desmin in the smooth muscle homogenate (lane G) as well as the purified

desmin control (lane 2). The anti-desmin also labels desmin that has bound to:

(a) synemin present in the smooth muscle homogenate (lane G), (b) purified

synemin (lane 1), (c) purified a-actinin (lane 3), and (d) the 55 kD fragment of a-actinin (lane 4). Desmin binding to the small amount of a-actinin in lane G

was just at the detection limit, and could be detected if the gel was overloaded.

A Western blot of a duplicate gel to that shown in Figure 5A (except without the 55 kD a-actinin fragment), overlayed with a-actinin and incubated with monoclonal anti-a-actinin antibodies and then with goat-anti-mouse IgM- horseradish peroxidase, is shown in Figure 5D. As expected, the antibodies label a-actinin present in the smooth muscle homogenate (lane G) as well as the purified a-actinin control (lane 3). The anti-a-actinin also labels a-actinin that has bound to: (a) desmin present in the smooth muscle homogenate (lane G), 150 (b) purified synemin (lane 1), and (c) purified desmin (lane 2). Although a trace

of binding of a-actinin to synemin in lane G is evident in this example, such binding was not always observed because of the small amount of synemin present in this sample. 151 DISCUSSION

We have shown in this study that synemin interacts with desmin in vitro by

using two different approaches. In the first one, the interaction was detected

by examining the effect of synemin on desmin's ability to assemble into IPs by

negative staining electron microscopy (Fig. 2). Self-assembly into

"reconstituted" filaments is perhaps the best known property of IF proteins.

Addition of increasing amounts of synemin to a constant amount of desmin

prior to dialysis against an IF-assembly buffer progressively decreased desmin's

ability to form long, smooth-sided filaments of ~ 10 nm diameter. The

Immunogold labeling results shown in Figure 3 indicated that the synemin was

bound to the shortened desmin IFs, but did not show a definite axial periodicity

of labeling along the filament. That the synemin was associated with the

desmin assembly products also was indicated by the lack of unbound,

~globular synemin structures in the desmin/synemin mixtures (Fig. 2). Our

assembly studies were initiated by first mixing highly purified desmin and

synemin in 10 mM Tris-HCI, pH 8.5. We [Pang et al., 1983; Ip et al., 1985a] have shown previously that, in such buffers, desmin exists as tetrameric protofilaments (i.e., ~2-3 nm diameter by ~50 nm long species, which contain four 53 kD polypeptides for a "functional" molecular mass of ~212kD). We also have shown [S.R. Bilak et al., 1991] that, under these ionic conditions, synemin exists as spherical particles of ~ 11 nm diameter that contain two 230 kD polypeptides (i.e., a "functional" molecular mass of -460 kD). Thus, at a 152 1:1 ratio (w/w; desmin:synemin) the molar ratio of desmin molecules

(tetramers) to synemin molecules (dimers) is about two to one (see Fig. 2B).

Many very short ( - 50-70 nm), full-width (~10 nm) filaments were present

under these conditions. An interesting result noticed herein was that

measurement of filament lengths revealed the presence of size (length) categories (e.g., -0.11 //m, ~0.17 fjm) of the short filaments formed in the presence of synemin. Perhaps this result reflects an underlying principle of IF assembly/structure, but further attempts at interpretation may be more fruitful after the assembly process of IF proteins is better understood [see Ip et al.,

1985a,b; Aebi et al., 1988; Albers and Fuchs, 1989; Stewart et al., 1989;

Chou et al., 1990; Hisanaga et al., 1990; Potschka et al., 1990; Steinert,

1990; Hatzfeld and Weber, 1991]. The marked effect of synemin on desmin assembly that we observed does not agree with studies of Sandoval et al.

[1983]. Those workers stated that synemin showed no effect either on the rate or extent of desmin polymerization. They, however, did not show those results, nor describe how they were conducted, thus, making it difficult to make comparisons. Those workers did report that synemin binds to reconstituted desmin filaments. In that experiment, synemin was added to already assembled desmin IFs at an approximate ratio of 2:1 (desmin:synemin, w/w). Incubation of that mixture with polyclonal anti-synemin antibodies and a fluorescently-labeled secondary antibody showed a bright staining pattern by examination at the light microscope level [Sandoval et al., 1983]. With that 153 level of resolution, however, it seems possible that the reactive synemin was

nonspecifically adhering to the glass coverslip, or was entrapped by the network of desmin filaments, rather than bound specifically to the desmin filaments. In our studies, we found much less evidence of synemin binding to fully-assembled desmin IPs than when synemin was present during the assembly process.

The second approach we used to examine desmin/synemin interaction entailed the use of solid-phase binding assays. We used both dot blot and gel blotting type methods. The dot blot has the advantage that the proteins are bound to the NC in non-denaturing solvents and have not been subjected to

SDS (and thus, may be somewhat more native), but can give erroneous false positive results if very highly purified proteins are not used [i.e., observed binding could be due to contaminant(s)]. The gel blotting method largely circumvents problems with contributions to binding due to impurities, but entails transfer of an SDS-denatured protein to the NC, which may be renatured to more variable degrees than with the dot blot method. Results of both assays indicated that overlaid synemin binds to desmin, and that overlaid desmin binds to synemin. These results were consistently obtained when the overlay steps were conducted in low ionic strength, slightly alkaline buffers (i.e., 10 mM Tris-

HCI, pH 8.5). Assays conducted under conditions where desmin prefers to assemble into IPs gave inconsistent results. Thus, our solid-phase binding results were in concert with our desmin/synemin assembly study (i.e., 154 interaction is favored when desmin is present as tetramers and synemin is

present as nonaggregated, -11 nm diameter spheres). Sandoval et al. [1983]

also reported that synemin interacts in vitro with soluble desmin. They found

that "soluble" synemin could effectively block {~ 65-70%) immunoprecipitation

of "soluble" desmin with anti-desmin antibodies. Again, however, detailed

comparison with our study is difficult. Examination of their procedure indicates

that the assays were conducted in a buffer at pH 7.4 containing 100 mM NaCI,

conditions in which desmin should be assembled into IPs [Huiatt, 1979; Huiatt et al., 1980; Ip et al., 1985a; Chou et al., 1990] and synemin already is aggregated into ~ 15-25 nm diameter, irregular complexes [S.R. Bilak et al.,

1991]. Thus, what the term "soluble" desmin meant in their study [Sandoval et al., 1983], is not clear.

A novel result obtained in this study is the binding between o-actinin and synemin and between a-actinin and desmin. Moreover, these interactions were observed when preparations of the 55 kD fragment of or-actinin, which contains the spectrin-like repeat rod domain (Arakawa et ai., 1985; Blanchard et al.,

1989; Davison et al., 1989), were utilized. The interactions we have shown, between any two members of the three proteins examined, also make it plausible that they act in a ternary complex. It is important to interpret results of any overlay assay with caution. Much depends upon successful retention of appropriate binding sites on proteins "plastered" on NC in the dot blot assays, and successful renaturation of binding sites in the proteins transferred to the 155 NC in the gel blotting assays; thus, failure to show binding is not conclusive.

False positives, especially in the dot blot assays, and even to some extent in

the gel blotting assays, also must be taken into consideration. Thus, all interactions shown by these methods will need to be examined in future studies employing more conventional biochemical assays.

In somewhat analogous reports to ours, interaction of desmin and spectrin has been described [Langley and Cohen, 1986, 1987], as has interaction of NF-

L with spectrin [Frappier et al., 1991]. Desmin has been shown also to bind to other proteins in vitro, including the IFAP, plectin [Foisner et al., 1988; Foisner and Wiche, 1991], ankyrin [Georgatos et al., 1987] and lamin B [Georgatos et al., 1987]. ff-Actinin also binds to F-actin [Robson et al., 1970], nebulin {[Nave et al., 1990]; presumably this entails only the C-terminal Z-llne domain of nebulin [Jin and Wang, 1991]}, integrin [Pavaiko and Burridge, 1991], and possibly [Beikin and Koteliansky, 1987]. In a detailed three-dimensional

TEM analysis of a-actinin in cultured cardiac muscle cells and nonmuscle cells,

Isobe et al. [1988] have suggested that a-actinin interlinks cytoskeletal elements, including IFs.

Recent studies in our laboratory have shown that desmin IFs surround the myofibrillar Z-lines in mature mammalian muscle cells, link adjacent myofibrils at their Z-line levels [M.M. Bilak et al., 1991b], and that synemin can either be considered part of, or attached to, these IFs at the Z-lines [M.M. Bilak, 1991a].

Evidence that IFs interact, structurally and biochemically, with myofibrillar Z-line 156 components would strengthen the role proposed for IPs in these cells, i.e., that

they link all myofibrils together [Lazarides, 1980; Richardson et al., 1981; M.M.

Bilak et al., 1991a] and, in turn, the myofibrils to other cellular structures [M.M.

Bilak et al., 1991a]. In this study we have presented biochemical support for this role. 157 ACKNOWLEDGMENTS

We thank Mary Sue Mayes for technical assistance. This work was supported, in part, by grants from the Muscular Dystrophy Association, USDA

Competitive Grants Program (Award 89-37265-4441), National Live Stock and

Meat Board, and the American Heart Association, Iowa Affiliate. This is Journal

Paper J-14738 of the Iowa Agriculture and Home Economics Experiment

Station, Ames, lA 50011, projects 2921, 2930, and 2127. 158 REFERENCES

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The purposes of the first section of my study were to: (1 ) examine selected

properties of synemin, and (2) identify synemin (or a synemin homolog) in

mammalian muscles. An integrated immunological, biochemical, and structural

approach was utilized in these experiments. The major conclusions reached

based upon results obtained in this section include the following:

(1) Synemin can be reliably purified to satisfactory homogeneity by

modification of a procedure described by Sandoval et al. (1983).

(2) Removal of urea from chromatographically purified synemin by dialysis against 10 mM Tris-HCI, pH 8.5, results in a soluble protein. Purified synemin in 10 mM Tris-HCI, pH 8.5, consists primarily of spherical particles (10.8 ±

0.15 nm) (mean±standard error of the mean) as shown by negative staining electron microscopy.

(3) Chemical crosslinking indicates that purified synemin exists primarily as a dimeric molecule in low ionic strength, slightly akaline buffers.

(4) Synemin's solubility properties are similar to those of desmin when the respective proteins are dialyzed against solutions of various pH values (with constant ionic strength) or against solutions of various ionic strengths (with constant pH). However, under ~ physiological-like conditions in which purified desmin self-assembles into long IPs, synemin tends to self-associate into complex (irregular, but overall ~ 15-25 nm diameter globular-like) aggregates, and is incapable of forming morphologically typical IPs under the conditions 166 tested. Synemin does form short, ~S-shaped structures - 10 nm in diameter

that are similar in appearance to those reported for NF-H, under conditions

peculiar for assembly of the NF triplet polypeptides into IFs.

(5) The presence of a synemin homolog in adult mammalian skeletal, cardiac,

and smooth muscle samples was demonstrated by Western blot analysis.

Whereas adult avian skeletal, cardiac, and smooth muscle samples contain a reactive 230 kD band, all three of the mammalian muscle samples contain two reactive bands (-225 kD and ~195 kD). Partially purified synemin isolated from porcine stomach smooth muscle also was enriched in the ~ 225 kD and

~195 kD bands when examined by SDS-PAGE, and these two bands were labeled with anti-avian synemin antibodies by Western blot analysis.

(6) Conventional immunofluorescence and confocal scanning laser microscopy of isolated myofibrils and of frozen sections of adult mammalian skeletal, cardiac, and smooth muscle samples showed that synemin is present inside all three muscle cell types. Synemin is colocalized with desmin at the myofibrillar Z-lines of adult mammalian and avian skeletal and cardiac muscle samples. In cryosections, synemin is present in a wavy filamentous pattern throughout adult mammalian smooth muscle cells.

The purpose of the second section of my study was to determine if synemin interacts with proteins located at or near the myofibrillar Z-lines, especially desmin, the major IF protein in adult striated muscle cells. The major conclusions obtained from the results in this section were as follows: 167 (1) Synemin specifically affects the ability of desmin to assemble into IPs.

Increasing the amount of synemin relative to that of desmin progressively

decreased the ability of desmin to assemble into long IPs. At a 5:1 ratio

(desminrsynemin, w/w), the filaments were generally short, with many of them

falling into size (length) categories of ~0.39± 0.007 //m, 0.17 ± 0.002 fjtm, or

0.11 ± 0.001 /ym (mean ±standard error of the mean). The number of very

short ( ~ 50-70 nm) filaments progressively increases as the amount of synemin

relative to desmin increases. These ~ 50-70 nm long filaments appear to be

the shortest stable -10 nm diameter filaments present. The longer size classes

of filaments formed in the presence of synemin may be multimers of these. On-

grid immunogold labeling of desmin/synemin mixtures with polyclonal anti-

synemin antibodies indicates that synemin was present both along the desmin

IPs and at points of IP intersection, which, in turn, indicates that synemin either

forms copolymeric IPs with desmin, or binds along the length of desmin IPs.

(2) Solid-phase binding assays, using both Western blot (electrophoretically- transferred proteins) and dot blot (spotted, non-denatured proteins) methods, show that synemin can bind to both desmin, the major protein of muscle IPs, and a-actinin, a major integral Z-line protein.

The results of both sections of my studies, taken in toto. indicate that the

IPAP synemin is ubiquitous to avian and mammalian muscle cells, show that synemin alters desmin IP assembly dynamics, and are consistent with the idea 168 that synemin may serve as a cytoskeletal crosslinking component between IPs and myofibrils in muscle cells. 169 LITERATURE CITED

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I wish to thank the members of the Muscle Biology Group, especially Mary

Bremner and Marlene Bright, for their expert technical assistance and advice. I would also like to especially thank Drs. Ted W. Hulatt and Marvin H. Stromer for their advice and comments. I extend thanks to Drs. Donald C. Beitz and

Donald C. Dyer for their advice and suggestions while serving on my committee. I especially thank my major professor, Dr. Richard M. Robson, for his advice, knowledge, valuable research discussions, and time during the course of this investigation. I would like to thank my parents and family for their support and patience. My deepest expression of appreciation Is for

Masako Maria Bilak, who has supplemented my graduate experience with love, patience, understanding, and support.