A study of invertebrate actins by isoelectric focusing and immunodiffusion

Couet, H. Gert de; Mazander, K.D.; Gröschel‐Stewart, Ute

doi:10.1007/bf01975112

Cited by 11 publications

(1 citation statement)

References 13 publications

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“…In the photoreceptor membrane of invertebrates, a number of nucleotide processing enzymes involved in phototransduction can be assumed to be present and partly responsible for this effect. We emphasize the importance of this observation because a number of structural and functional studies (23,31,60) did not recognize the need for actin stabilizing conditions if the cytoskeleton is to survive . Incubation of rhabdomeral membranes in the presence of Cat + for 4 h at room temperature gave no evidence of any proteolytic activity present in the sample that could be responsible for the breakdown of cytoskeletal elements (not shown).…”

Section: Inhibition Of Dnase I Decays With Timementioning

confidence: 92%

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

Couet¹,

Stowe²,

Blest³

1984

The Journal of Cell Biology

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Infiltration of compound eyes of crayfish, Cherax destructor, with the thiol protease inhibitor Ep-475 or with trifluoperazine prior to fixation for electron microscopy was found to stabilize an axial filament of 6-12 nm diam within each rhabdomeral microvillus of the photoreceptors . Rhabdoms isolated from retinal homogenates by sucrose gradient centrifugation under conditions that stabilize cytoskeletal material contained large amounts of a 42-kd polypeptide that co-migrated with insect flight muscle actin in one-and two-dimensional PAGE, inhibited pancreatic DNase I, and bound to vertebrate myosin . Vertebrate skeletal muscle actin added to retinal homogenates did not co-purify with rhabdoms, implying that actin was not a contaminant from nonmembranous structures. DNase I inhibition assays of detergent-lysed rhabdoms indicated the presence of large amounts of filamentous actin provided ATP was present . Monomeric actin in such preparations was completely polymerizable only after 90 min incubation with equimolar phalloidin . More than half of the actin present could be liberated from the membrane by sonication, indicating a loose association with the membrane . However, a large proportion of the actin was tightly bound to the rhabdomeral membrane, and washing sonicated membrane fractions with solutions of a range of ionic strengths and nonionic detergents failed to remove it. Antibodies to scallop actin only bound to frozen sections of rhabdoms after gentle permeabilization and very long incubation periods, probably because of steric hindrance and the hydrophobicity of the structure . The F-actin probe nitrobenzoxadiazol phallacidin bound to rhabdoms and labeled F-actin aggregates in other retinal components, but rhabdom fluorescence was not abolished by preincubation with phalloidin. The biochemical data indicate the existence of two distinct actin-based cytoskeletal systems, one being closely membrane associated. The other may possibly constitute the axial filament, although the evidence for this is equivocal .The rhabdomeres of arthropod and cephalopod photoreceptors are composed of numerous microvilli which in a given cell-type are remarkably uniform in diameter and ordered in arrays ofalmost crystalline regularity . Successive authors have implied or suggested that a cytoskeleton is likely to underlie this organization (6,31,37,59,60,65), and some have speculated that it will prove to be actin based. Actin is a component of rhabdoms of squid (59, 60), and the organization ofother microvillar types provides well known precedents (50,54,68).Conventional techniques of electron microscopy have not revealed a cytoskeletal organization within rhabdomeral microvilli with any consistency, and an early demonstration of an axial filament in microvilli of bee photoreceptors (70) has been largely overlooked in the literature. Recently, three studies have demonstrated an axial filament linked to the plasma membrane by side-arms in photoreceptor microvilli offlies (7, 8) and squid (60). Although these filament...

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Section: Inhibition Of Dnase I Decays With Timementioning

confidence: 92%

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

Couet¹,

Stowe²,

Blest³

1984

The Journal of Cell Biology

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Actin in cellular components of the basement membrane of the compound eye of a blowfly

Blest

Couet

1983

Cell Tissue Res.

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The so-called 'basement membrane' of arthropod compound eyes is known to be of heterogeneous origin (Odselius and Eloffson 1981). A major contribution in Diptera with open rhabdoms is provided by a pigmented component which lies at the basal end of the extracellular space of each ommatidium and fills it, the glial plug. Ancillary components consist of the expanded tips of cone cell processes. Each glial plug exhibits two distinct regions: ramifying processes extend into the extracellular space and contain numerous pigment granules, while proximally the cytoplasm is devoid of granules but packed with bundles of cross-linked microfilaments that bind the fluorescent F-actin probe NBD-phallacidin strongly and antibodies to scallop actin weakly. Cone cell expansions also contain microfilaments and exhibit the same binding properties. The proximal faces of the cells of the glial plugs and of the cone cell expansions are covered with a coarsely fibrillar extracellular matrix. Some actin bundles appear to be attached to the plasma membranes at their ends, although the reality of this arrangement is still in question. Cellular components of the basement membrane are bonded together by their extracellular matrices, so that collectively they provide a reinforced network that retains the retina. Bundles of axons from the photoreceptors and tracheae that supply the retina with tracheoles pass through the spaces in this network.

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Studies on the antigenic sites of actin: a comparative study of the immunogenic crossreactivity of invertebrate actins

1983

J Muscle Res Cell Motil

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The structural homologies of invertebrate actins with cytoplasmic vertebrate actins have recently been substantiated by comparative sequence analyses. This suggests that cytoplasmic actin is the ancestral precursor of smooth and striated muscle actin in vertebrates. We have raised antibodies in rabbits against a number of invertebrate muscle actins and have characterized the antisera by means of the highly sensitive ELISA method, which allows quantitation of nanomolar amounts of actin. Despite the fact that the invertebrate actins examined are very similar in primary structure, our results indicate that antibodies raised against them clearly distinguish between only a few amino-acid substitutions, and that the immunoreactivities quantitatively reflect the genetic divergence of this ubiquitous conservative protein. Examination of several proteolytic fragments of scallop actin for immunoreactivity with the homologous antiserum suggests that the major antigenic sites of actin are located within the amino terminal region of the molecule, while a carboxy terminal fragment comprising residues 69-372 exhibits very weak crossreactivity. Immunoadsorption experiments further indicate that species-specific antibodies are directed to antigenic determinants in the N-terminal region. This finding is supported by an examination of the effects of chemical modifications to Tyr, His, Arg, and Cys residues on the immunoreactivity of actin. Interaction with DNAase I markedly decreases the immunoreactivity of actin. This is consonant with the finding that the amino terminal peptide comprising residues 1-207 inhibits DNAase I, whilst a tryptic fragment fails to bind to the enzyme. The interaction is abolished by EDTA and the removal of the tightly bound cation is accompanied by a conformational change, shown by shifts in circular dichroic spectra. The possible involvement of the amino terminal peptide of actin in cation binding is discussed.

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A study of invertebrate actins by isoelectric focusing and immunodiffusion

Abstract: Actin isolated from various invertebrate phyla comigrates with the beta-form of vertebrate smooth muscle actin. However, invertebrate actins are not identical, since antibodies to insect-actin will not crossreact with the other species.

Cited by 11 publications

References 13 publications

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

Actin in cellular components of the basement membrane of the compound eye of a blowfly

Studies on the antigenic sites of actin: a comparative study of the immunogenic crossreactivity of invertebrate actins

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