UVM Theses and Dissertations
Format:
Print
Author:
Alvarez-Ortiz, Pedro
Dept./Program:
Biology
Year:
2014
Degree:
Ph. D.
Abstract:
Muscle and basement membrane (BM) are two distinct features that are conserved throughout the Kingdom Animalia. Striated muscle is characterized by a repertoire of conserved proteins that include actin and myosin, in addition to other proteins that together make the sarcomere, the basic unit of contraction that is repeated along the length of a myofibril. The muscle, in order to transmit its contractile force, requires connections between myofibrils and the extracellular environment. These connections occur via the dystrophin-glycoprotein complex that links the muscle to the surrounding extracellular BM. BM is composed of 10 conserved proteins: collagen IV, laminin, perlecan, and other proteins that together form a meshwork that surrounds muscle. This dissertation is focused on the phylogeny and expression patterns of two Drosophila proteins, flightin and glutactin: flightin is a sarcomeric protein essential in the indirect flight muscle while glutactin is an extracellular BM protein previously shown to surround muscle during embryonic development.
We determined the phylogeny of glutactin and flightin using Blast to determine how widespread these proteins are in the Kingdom Animalia. For glutactin, we characterized its expression pattern during larva and adult stages by immunostaining and confocal microscopy to identify the tissues and organs where glutactin is present. Also, by using RNA-interference we down-regulated glutactin and tested its effect in the larva and adult by using locomotory (larval crawling and adult flight) and ovoposition assays. To study the expression patterns of flightin we used RT-PCR to test larva and adults from different species of basal insects as well as derived . species from dipterans. Our phylogenetic results from glutactin and flightin revealed that both proteins are taxonomically restricted: glutactin is present only in the Drosophila group whereas flightin is confined to Pancrustacea. The expression pattern of glutactin in the larva revealed that in addition to being a BM protein surrounding the body wall muscles, glutactin is also a muscle protein present in the costameres. Its down-regulation in the muscles of the larva slowed their crawling speed in comparison to controls.
In adults, glutactin was detected surrounding direct flight muscle, the abdominal intestinal tract and reproductive system. Its ubiquitous down-regulation revealed that more than 50 percent of the adult flies died during the first two to three days after eclosion, and the surviving adult have reduced lifespans. In addition, the number of embryos laid by females that survived past three days was significantly reduced compared to control females. Result from the expression of flightin in different species of insects revealed that it's broadly expressed except in those Diptera species that belong to the SchizQphora lineage where its expression is limited to the thorax. Moreover, we detected the presence of flightin isoforms in the larva of several species that are different from corresponding flightin isoform detected in the adult. Results from this study suggest that taxonomic restricted proteins play important roles in the organism because their absence or down-regulation demonstrated the loss of features that seems essential for normal life.
We determined the phylogeny of glutactin and flightin using Blast to determine how widespread these proteins are in the Kingdom Animalia. For glutactin, we characterized its expression pattern during larva and adult stages by immunostaining and confocal microscopy to identify the tissues and organs where glutactin is present. Also, by using RNA-interference we down-regulated glutactin and tested its effect in the larva and adult by using locomotory (larval crawling and adult flight) and ovoposition assays. To study the expression patterns of flightin we used RT-PCR to test larva and adults from different species of basal insects as well as derived . species from dipterans. Our phylogenetic results from glutactin and flightin revealed that both proteins are taxonomically restricted: glutactin is present only in the Drosophila group whereas flightin is confined to Pancrustacea. The expression pattern of glutactin in the larva revealed that in addition to being a BM protein surrounding the body wall muscles, glutactin is also a muscle protein present in the costameres. Its down-regulation in the muscles of the larva slowed their crawling speed in comparison to controls.
In adults, glutactin was detected surrounding direct flight muscle, the abdominal intestinal tract and reproductive system. Its ubiquitous down-regulation revealed that more than 50 percent of the adult flies died during the first two to three days after eclosion, and the surviving adult have reduced lifespans. In addition, the number of embryos laid by females that survived past three days was significantly reduced compared to control females. Result from the expression of flightin in different species of insects revealed that it's broadly expressed except in those Diptera species that belong to the SchizQphora lineage where its expression is limited to the thorax. Moreover, we detected the presence of flightin isoforms in the larva of several species that are different from corresponding flightin isoform detected in the adult. Results from this study suggest that taxonomic restricted proteins play important roles in the organism because their absence or down-regulation demonstrated the loss of features that seems essential for normal life.