Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50

Abstract

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation.KEYWORDS microtubule motors; endocytosis; cell polarity; kinesin; dynactin M ICROTUBULE motors such as cytoplasmic Dynein (hereafter referred to as Dynein) and proteins of the Kinesin superfamily play essential roles in cargo transport. Dynein is a minus-end motor and is responsible for the majority of minus-end transport within the cell (Kardon and Vale 2009). Mammalian genomes encode .40 different Kinesins, and most of these move cargo toward the plus-end of microtubules (Hirokawa et al. 2009). One type of cargo that is known to be transported by microtubule motors are vesicles of the endolysosomal system.Cargoes that enter the cell via endocytosis follow numerous sorting pathways that ultimately determine their fate. For example, nutrient receptors such as the Transferrin receptor, are recycled back to the plasma membrane (Mayor et al. 1993;Huotari and Helenius 2011). Growth factor receptors and signaling molecules are often targeted for degradation (Beguinot et al. 1984;Huotari and Helenius 2011). This is necessary to attenuate growth-promoting signals. Persistent and uncontrolled growth-promoting signals are associated with cancer (Normanno et al. 2006). Cargoes that are destined for degradation transit through vesicles that undergo maturation from early endosome to late endosome. Late endosomes eventually fuse with acidic, degradative organelles known as lysosomes. Endocytic maturation involves the progressive and ordered association of specifi...

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