Molecular and cellular mechanisms of tracheal invasion of polarized muscle membrane networks in Drosophila [electronic resource]

From a structural standpoint, one of the most characteristic general design elements of the mammalian organism is its tubular nature. The lung and circulatory system shuttle oxygen and nutrients to target tissues and allow for the excretion of waste products. In some ways, the vasculature lies at the center of human physiology--its passageways provide the infrastructure for maintaining homeostasis. Despite the importance of tubular networks in human health and disease, we have a poor understanding of many aspects of the genetic, molecular, and cellular programs controlling the development of these complex structures. The Drosophila melanogaster tracheal system, an elaborate network of hollow epithelial tubes, transports gases to and from target tissues. The tracheal system, with its simple structure, tractable genetics, and substantial experimental toolkit has emerged as an excellent model system for studying questions with relevance to more complex tubular systems. During development, tracheal branches ramify on the surface of target tissues, providing oxygen to every cell in the body. However, in a phenomenon unique to the Drosophila flight muscle, trachea are also present within plasma membrane invaginations deep below the muscle's outer extremities and have been described to surround every mitochondria of the flight muscle, thus coupling oxygen delivery directly to aerobic respiration at the mitochondrion. Although the presence of trachea within flight muscle membrane invaginations has been described for over 150 years, the developmental progression and cellular and molecular basis of this subcellular targeting process is unknown. In Chapter 2, we show that tracheal branches invade the developing flight muscle Transverse (T)-tubule plasma membrane invagination system during a brief period of pupal development. Branchless (Bnl) FGF, a fibroblast growth factor that functions as a chemoattractant, is required in the flight muscle, and its cognate receptor, Breathless (Btl) FGFR, is required in trachea for the tracheal invasion process to occur. Whereas Bnl FGF is localized to all flight muscle plasma membranes prior to tracheal invasion, during invasion Bnl FGF localizes preferentially to the T-tubule and is excluded from the surrounding plasma membrane. In addition to Bnl FGF, core polarity regulators commonly found on basolateral membranes in epithelial cells also preferentially localize to the T-tubule network during tracheal invasion. We find that depletion of AP-1[gamma], targeting machinery required for basolateral secretion in Drosophila epithelia, can also reroute Bnl FGF secretion to the outer plasma membrane and away from T-tubule openings, shifting trachea to the plasma membrane and away from T-tubules. We propose that (1) polarized secretion of Bnl FGF to the T-tubule guides tracheal branches into the T-tubule network and that (2) polarized Bnl FGF secretion is established via the redeployment of ancestral basolateral secretion pathways to the T-tubules, a membrane domain having molecular signatures of epithelial basolateral domains. To our knowledge, compartmentalized secretion of Bnl FGF to flight muscle T-tubule membranes is the first example of polarized subcellular secretion of a growth factor with functional consequences for the development of another tissue. In Chapter 3, we examine the molecular machinery involved in maintaining the polarized secretion of Bnl FGF during tracheal invasion. We find a host of secretory machinery, including several Rabs, Myosin V, an actin nucleator, and others, to be involved in the secretion of Bnl-FGF to the T-tubule during tracheal invasion. From these data we propose a molecular model to explain polarized Bnl FGF secretion at the T-tubule. Why is the flight muscle the only tissue invaded by trachea? In Chapter 4, we find that the flight muscle is structurally adapted to allow its T-tubule plasma membrane invagination network to be co-opted by migrating tracheal processes. In contrast to other muscles, the flight muscle T-tubule network forms large holes on the muscle surface as part of its development. The tracheal invasion process in the flight muscle is therefore the consequence not only of a polarized secretion process as discussed in Chapter 2, but also a structurally distinct stage of flight muscle development. In the final chapter, we search for the fine tracheal tubes reported to target and form contacts on the flight muscle mitochondria. We find an extracellular protein-based lattice of the appropriate diameter and localization to potentially represent the reported tracheal extensions to the mitochondria and propose several models of protein lattice formation. We hope that insight derived from this work spurs future investigators in a host of areas. The exquisite example of targeting to specific membrane domains dependent on a subcellular chemoattractant gradient demonstrated by the invading tracheal branch may provide insight into a number of developmental scenarios where fine targeting of different cells is required. We also hope that our finding regarding polarized secretion in muscle inspires new ways to think about the assembly of muscle membranes during development and disease states.

Keywords: Biochemistry.
Advisor: Krasnow Mark
Brown Patrick O'Reilly
Luo Liqun
Straight Aaron