EPI64 regulates microvillar subdomains and structure

EPI64 is a TBC domain–containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

Plasma membranes from insect midgut cells

Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modified into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffic inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifications are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane) observed in the midgut cells of hemipterans (aphids and bugs). The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content) that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.

The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis.

Photoreceptor cells of the Drosophila compound eye begin to develop specialized membrane foldings at the apical surface in midpupation. The microvillar structure ultimately forms the rhabdomere, an actin-rich light-gathering organelle with a characteristic shape and morphology. In a P-element transposition screen, we isolated mutations in a gene, bifocal (bif), which is required for the development of normal rhabdomeres. The morphological defects seen in bif mutant animals, in which the distinct contact domains established by the newly formed rhabdomeres are abnormal, first become apparent during midpupal development. The later defects seen in the mutant adult R cells are more dramatic, with the rhabdomeres enlarged, elongated, and frequently split. bif encodes a novel putative protein of 1063 amino acids which is expressed in the embryo and the larval eye imaginal disc in a pattern identical to that of F actin. During pupal development, Bif localizes to the base of the filamentous actin associated with the forming rhabdomeres along one side of the differentiating R cells. On the basis of its subcellular localization and loss-of-function phenotype, we discuss possible roles of Bif in photoreceptor morphogenesis.

Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury

The association/dissociation of ezrin, a microvillar membrane-cytoskeleton linker, was studied to search for the initial step leading to anoxia-induced brush-border breakdown in a rabbit proximal tubule suspension. Electron microscopy studies display time-dependent damage to the microvilli during anoxia; immunoblots demonstrate the dissociation of ezrin from the cytoskeleton, reflected by the significant decrease in Triton X-100-insoluble ezrin from control (91%) to 39% after 30 min. Simultaneously, Triton X-100-soluble and extracellular ezrin increased with no change in total ezrin, Triton X-100 solubility of actin, or total intracellular protein. Parallel immunocytochemistry studies show diffusion of ezrin from the brush border, where ezrin is highly colocalized with F-actin during normoxia into the cytoplasm. Thirty minutes of reoxygenation following 30 min of anoxia causes recovery of the microvillar structure and reassociation of ezrin to the cytoskeleton and the brush border. Application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) or inhibition of intracellular calpain or calcineurin do not prevent the dissociation of ezrin during anoxia. We conclude that ezrin-cytoskeletal dissociation may initiate microvillar breakdown during anoxia via calcium-independent mechanisms.

Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells.

The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.