Spectrin-actin associations studied by electron microscopy of shadowed preparations

Cohen, Carl M.; Tyler, J M; Branton, Daniel

doi:10.1016/0092-8674(80)90451-1

Cited by 156 publications

(63 citation statements)

References 21 publications

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“…The proximity of the ankyrin-binding site to the spectrin self-association site has also been observed by electron microscopy [36, 371. Based on the analogy with c1 actinin, and the similarity of the carboxy-terminus of c1 spectrin and the amino-terminus of / i ' spectrin, it seems likely that this is the region of spectrin that is involved in actin binding [34]. This hypothesis is consistent with earlier electron microscopy observations that indicated that protein 4.1 [37] and filaments of F-actin [38] both bind at the tail end of spectrin, opposite the self-association site. In vitro binding studies with hybrid spectrins have also demonstrated that the p subunit is responsible for bestowing protein-4.1 sensitivity on the spectrin-actin interaction [39].…”

supporting

confidence: 81%

Radiolabel‐transfer cross‐linking demonstrates that protein 4.1 binds to the N‐terminal region of β spectrin and to actin in binary interactions

Becker

Schwartz

Morrow

et al. 1990

European Journal of Biochemistry

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Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, l-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of j spectrin within a sequence homologous to the actinbinding region of c1 actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4. I-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.The erythrocyte membrane skeleton is composed primarily of spectrin, actin, proteins 4.1, 4.9 and adducin. The skeleton is attached to the lipid bilayer by spectrin's interaction with ankyrin (syndcin; protein 2.1) [l -41 which binds to the transmembrane protein 3 [5. 61. A second membrane attachment occurs via spcctrin's interaction with protein 4.1, which in turn binds to the transmembrane glycoproteins, glycophorin A [7], glycophorin C [S] and protein 3 [9]. Spectrin is a highly elongated heterodimer, composed of intertwined c1 and p chains (proteins 1 and 2, respectively), aligned in antiparallel fashion with respect to the amino-termini. Spectrin dimers self-associate head-to-head to form tetramers and higher oligomers [ 10 -121. Spectrin also binds to F-actin [ 13 -151 and this interaction is greatly enhanced by protein 4.1 [16][17][18][19]. Protein 4.9 [20] and adducin [21, 22) may serve as actinbundling proteins and the latter protein also enhances spectrin-actin binding.Spectrin structure was initially defined as a linear sequence of large peptides, called domains, separated by protease-sensitive regions [23, 241. Later studies determined that spectrin consisted of a series of homologous 106-amino-acid repeats arranged in triple ci helices [25]. When the cDNA for spectrin was isolated and sequenced, it became evident that spectrin is a member of a supergene family that includes c1 actinin and dystrophin [26 -321. The amino-terminal region of p spectrin and the carboxy-terminal region of ci spectrin contain nonrepetitive sequence with a high degree of similarity to the amino-and carboxy-terminal regions of the actin-binding protein, a actinin [33, 341. The specific sites on spectrin responsible for binding ankyrin or for self-association have been localized to particular proteolytic peptides by in vitro binding studies [35], and these sites are located near the 'head' corresponding to the Nterminal c1 chain/C-terminal p chain...

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supporting

confidence: 81%

Radiolabel‐transfer cross‐linking demonstrates that protein 4.1 binds to the N‐terminal region of β spectrin and to actin in binary interactions

Becker

Schwartz

Morrow

et al. 1990

European Journal of Biochemistry

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“…In our observations, actin was associated with the erythrocyte membrane only in the F-form through the filamentous components . The mode of such association on the membrane apparently resembles that of the spectrin-actin interaction in vitro (10) .…”

Section: Discussionmentioning

confidence: 99%

“…F-actin can bind to the Spectrin-reassociated membranes (9,10) . Detailed ultrastructural study of such reassociation may lead to better understanding of the organization and function of the cytoskeleton .…”

mentioning

confidence: 99%

Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

Tsukita¹,

Ishikawa²,

Sato³

et al. 1981

The Journal of Cell Biology

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Reassociation of spectrin and actin with human erythrocyte membranes was studied by stereoscopic electron microscopy of thin sections combined with tannic acid-glutaraldehyde fixation . Treatment of the erythrocyte membrane with 0.1 mM EDTA (pH 8.0) extracted >90% of the spectrin and actin and concomitantly removed filamentous meshworks underlying the membranes, followed by fragmentation into small inside-out vesicles . When such spectrindepleted vesicles were incubated with the EDTA extract (crude spectrin), a filamentous meshwork, similar to those of the original membranes, was reformed on the cytoplasmic surface of the vesicles . The filamentous components, with a uniform thickness of 9 nm, took a tortuous course and joined one another often in an end-to-end fashion to form an irregular but continuous meshwork parallel to the membrane . Purified spectrin was also reassociated with the vesicles in a population density of filamentous components almost comparable to that of the crude spectrin-reassociated vesicles . However, the meshwork formation was much smaller in extent, showing many independent filamentous components closely applied to the vesicle surface. When muscle G-actin was added to the crude spectrin-or purified spectrinreassociated vesicles under conditions which favor actin polymerization, actin filaments were seen to attach to the vesicles through the filamentous components . Two modes of association of actin filaments with the membrane were seen : end-to-membrane and side-to-membrane associations . In the end-to-membrane association, each actin filament was bound with several filamentous components exhibiting a spiderlike configuration, which was considered to be the unit of the filamentous meshwork of the original erythrocyte membrane .The existence of the cytoskeletal network underlying the erythrocyte membrane is now well documented (for reviews, see references 23, 25, 42). Evidence has accumulated that the erythrocyte cytoskeleton is mainly constituted of spectrin, the major peripheral membrane protein (28,47,48) . It is reasonably proposed that the erythrocyte cytoskeleton may play an important role in regulating the topography of intramembranous proteins (11,12,29,31,38) and in determining erythrocyte shape and deformity (4,16,27,35). In search of the supramolecular organization of the cytoskeleton, extensive studies have recently been directed to the structure (18,33,39) and chemical nature of spectrin (15,24,25,41), including its binding with other proteins, such as erythrocyte actin, band 2 .1, and band 4 .1 (2,14,22,45,47,49) . Although the erythrocyte actin accompanied spectrin during extraction with low-salt EDTA, 70 SACHIKO TSUKITA, SHOICHIRO TSUKITA, HARUNORI ISHIKAWA, SHINGO SATO, and MAKOTO NAKAO

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“…However, connectin binding to the side of a filament near the barbed end also could account for such an appearance. Indeed, spectrin and vinculin have been shown to bind to the sides of actin filaments (13,31). These cytoplasmic proteins have been postulated to be involved in the attachment of actin filaments to membranes.…”

Section: Discussionmentioning

confidence: 99%

Connectin: cell surface protein that binds both laminin and actin.

Brown¹,

Malinoff²,

Wicha³

1983

Proc. Natl. Acad. Sci. U.S.A.

114

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A purified cell surface receptor protein for laminin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high affinity. This binding was demonstrated both by cosedimentation of the receptor with actin and binding of the receptor to actin immobilized on nitrocellulose filters. Specificity was demonstrated by displacement of 'S-labeled receptor by unlabeled receptor. Scatchard analysis of receptor binding to actin yielded a Kd of 6 x 10-7 M. The receptor was observed to reduce the viscosity of actin filaments. It also caused the formation of bundles of parallel filaments. This observation and the stoichiometry of binding suggest that the receptor binds along the sides of actin filaments. Based on the ability of this receptor to bind both extracellular laminin and intracellular actin, we have named this protein "connectin." Connectin may be an example of a transmembrane protein that is capable of mediating the interaction of a cell with its extracellular matrix.

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Spectrin-actin associations studied by electron microscopy of shadowed preparations

Cited by 156 publications

References 21 publications

Radiolabel‐transfer cross‐linking demonstrates that protein 4.1 binds to the N‐terminal region of β spectrin and to actin in binary interactions

Radiolabel‐transfer cross‐linking demonstrates that protein 4.1 binds to the N‐terminal region of β spectrin and to actin in binary interactions

Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

Connectin: cell surface protein that binds both laminin and actin.

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