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

Abstract

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...

Feet, bridges, and pillars in triad junctions of mammalian skeletal muscle: Their possible relationship to calcium buffers in terminal cisternae and T-tubules and to excitation-contraction coupling

“…This was achieved by (i) blocking calcium-activated proteases with Ep-475 (Blest, Stowe & Eddey, 1982;de Couet, Stowe & Blest, 1984), (ii) by light tannic acid fixation (Stowe, Fukudome & Tanaka, 1986), and (iii) by reducing osmium concentration and exposure time during osmication (Stowe et al, 1986).…”

The molecular basis of mechanisms underlying polarization vision

“…Several studies have previously suggested that simply a restricted orientation and decreased mobility of the visual pigment within photoreceptor membranes is the mechanism for increased PS, particularly in rhabdomeric photoreceptors. The suggested mechanisms include decreased microvillar membrane fluidity [32,52] and channelling of opsin molecules by membrane-associated barriers, such as integral membrane glycoproteins, to reduce translational diffusion [51,55,82]. However, on its own, a simple change in fluidity, i.e.…”

“…Early studies of cephalopod photoreceptor structure reveal that the microvilli contain a cytoplasmic core bundle of actin filaments with crossbridges to the membranes, as well as special membrane junctions linking adjoining microvilli [53]. Microvillar actin cores have also been characterized in Drosophila and crayfish rhabdoms [54,55]. The observed actin cores and the core-to-membrane crossbridges fit with the idea from studies of Drosophila phototransduction of a 'signalplex'-a macromolecular complex linking the visual pigment and associated phototransduction proteins with the cytoskeleton (figure 3; for review see [56]).…”

Actin depolymerization is not involved in phototransduction

“…The rhabdomeral microvilli in the eyes of invertebrates have a cytoskeletal core (Saibil, 1982;Blest et al, 1982o,6, 1983) of actin (de Couet et al, 1984;Arikawa et al, 1990;Williams, 1991;Caiman & Chamberlain, 1992) which might be involved in phototransduction. Tsukita et al (1988) suggested that light stimulation triggers the breakdown of the microvillar actin filament complex and that this may play a role in phototransduction.…”