An Analysis of the Spatial, Temporal, and Functional Role of Key Proteins in the Drosophila Melanogaster Salivary Gland During Exocrine Secretion

Author

Kathryn Lantz, University of Nevada, Las VegasFollow

Award Date

12-1-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Committee Member

Andrew J. Andres

Second Committee Member

Laurel Raftery

Third Committee Member

Allen Gibbs

Fourth Committee Member

Frank van Breukelen

Fifth Committee Member

Ernesto Able-Santos

Number of Pages

384

Abstract

For over 60 years, the larval salivary gland of Drosophila melanogaster has been the ultimate model for elucidating how steroid hormones trigger genome-wide changes in gene expression in a specific target tissue. Because of the unusual interphase chromosomes of the salivary gland, the regions of the genome that are being induced by the steroid/receptor complex actually form puffs that are easily visible in compound microscopy. Despite the importance of this system, little has been elucidated over the years regarding how the hormone exposure actually changes the physiological activity of this secretory system. The work presented in this dissertation makes a significant contribution to our understanding of the hormone response of this tissue at the molecular level. It is also likely that this information can be broadly applied to steroid-responsive exocrine tissues in more complex animals including humans.

The major function of the larval salivary gland is the production and eventual secretion of a mix of highly glycosylated mucin proteins called glue. Although both the synthesis and secretion of glue is regulated by the major steroid hormone in insects, 20-hydroxyecdysone (20E), my work is mostly focused on the 20E stimulated secretion of that glue that occurs at the end of the instar. Eight hours before puparium formation, glue proteins are packaged into dense-core granules that are retained within the cells until they are exposed to a high titer of 20E. Within an hour of the steroid pulse, calcium levels are elevated and the granules begin to become docked at the apical membrane to release their cargo into the lumen of the tissue. By filling in some key molecular details of this process, I have contributed to our general understanding of how exocrine tissues respond to steroids and secrete their cargoes.

To create a solid foundation for examining exocrine secretion in the salivary gland, I began by characterizing the structure and organization of the wild type salivary gland during its response to 20E. This temporal analysis using more than 50 fluorescently tagged marker proteins was performed in live salivary glands to generate snap shots at known developmental time points. The work was later extend using time-lapse imaging techniques to generate movies of the complete secretion process in real time. From these analyses I was able to divide the 20E-regulated physiological response of the gland into four stages: 1) Post 20E exposure prior to any secretion events, 2) early secretion events that occur when only small vesicles containing glue cargoes are dumped, 3) mid secretion events in which mature dense-core secretory granules are docked and dumped with the aid of the actinomysin complex, and 4) late secretion events in which many granules, unable to completely dump their cargo, reform as inclusions within the cell.

I chose to further focus on three well-known molecular families that drive secretory events: Rab GTPases, myosin motor proteins, and calcium transporters. Rab GTPases are needed to target intracellular vesicles and secretory granules; the myosins provide the mechanical force to move granules and slide actin filaments; whereas, calcium transporters are required for the rapid Ca2+ elevations needed to sustain the response. These protein families contain a large number of members, so the purpose of this study is to identify and define the function of the Rab GTPase(s), myosin motor(s), and calcium transporters that are regulated by 20E and are needed for the secretion of glue.

Lastly, I explored, to a lesser extent, the role of other molecular families suspected to be involved in exocrine secretion. These families included kinesins, dyneins, EF-hand containing proteins, and gap junction proteins. All these preliminary studies create a general hypothesis of some essential proteins and molecular pathways that are involved in 20E-regulated secretion.

In this dissertation, I have observed the role of Rab1 and Rab11 in the maturation of secretory granules. F-actin plays an essential role in the dumping of glue into the lumen through Facsin, an F-actin stabilizer that is anchored to the apical membrane through the 20E induced Rab35. The F-actin cage structures that form around docked granules are acted upon by Myosin II, which is composed of the 20E-induced Myosin heavy chain Zipper and activated by Rho1 and Rok through the Myosin light chain Sqh. Along with the F-actin cytoskeletal elements, microtubules are needed for secretion through the kinesin and dynein motor proteins with shot, a 20E-induced molecule, acting as a crosslinker between the F-actin and microtubular networks in granule hand off. Lastly, the transporters required for proper calcium signaling and membrane fusion proteins have been highlighted, through there induction by 20E remains to be uncovered. I have provided a general yet comprehensive outline of the necessary proteins involved in salivary gland exocrine secretion.

Disciplines

Biology

File Format

pdf

Degree Grantor

University of Nevada, Las Vegas

Language

English

Repository Citation

Lantz, Kathryn, "An Analysis of the Spatial, Temporal, and Functional Role of Key Proteins in the Drosophila Melanogaster Salivary Gland During Exocrine Secretion" (2017). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3146.
http://dx.doi.org/10.34917/11889715

Rights

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