Functional and Mechanistic Study of CapZ and FAM129B as Two Novel Regulators in Endolysosomal Trafficking


Student thesis: Doctoral Thesis

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Awarding Institution
  • Jianbo YUE (Supervisor)
Award date2 Feb 2021


Endocytosis is an evolutionarily-conserved cellular process, which comprises a series of dynamically-interconnected membrane-enclosed vesicular structures, including early endosomes, endosomal carrier vesicles, and late endosomes, and this process is important for maintaining cellular homeostasis and energy recycling. The invagination of endocytic vesicles, early endosome maturation and fusion, and the subsequent transition to a late endosome is a prerequisite for the normal function of endolysosomal trafficking. Actin filaments, which provide the force to bend the membrane, are involved in the internalization of the endocytic vesicles. It is also reported that the actin comet tails that form around endocytic vesicles, propel these vesicles away from the plasma membrane. Although the role of actin filaments in the internalization of endocytic vesicles and their subsequent release from the plasma membrane has already been documented, their role in the late stages of endosomal trafficking is poorly studied. The recruitment, activation, and inactivation of different Ras-associated binding (RAB) proteins are known to play an essential role in controlling the identity and maturation of endosomes. Like other small GTPases, RAB-GDP is inactive, whereas RAB-GTP is active, and the switch from the inactive form to the active form and vice versa is catalyzed by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively. Different RABs have their own specific GEFs and GAPs, and some RABs (e.g., RAB5) also contain intrinsic GTPase activity. During endolysosomal trafficking, RAB5 and RAB7 are the hallmarks of early and late endosomes to regulate their maturation, respectively. However, the detailed mechanism that underlies the temporal and spatial regulation of these two RABs remains elusive. In addition, the Mon1-CCZ1 complex-mediated displacement of the RAB5 GEF (Rabex-5) favors the transition of a RAB5 to a RAB7-positive endosome. However, the initial triggers of early-to-late endosome transition are still unknown.

The small chemical, vacuolin-1 (V1), was originally found to induce the large vacuole formation by promoting the homotypic fusion of endosomes or lysosomes. In chapter three, to discover novel regulators of endolysosomal trafficking, I synthesized a biotinylated version of V1(Biotin-V1), which exhibits similar bioactivity to V1. This Biotin-V1was then used as bait in HeLa cell extracts to capture any interacting proteins. After immunoprecipitation of the V1-binding complex with streptavidin beads, 84 potential V1-binding proteins were identified in three independent experiments with high fidelity by mass spectrometry. Among them, I found that CapZ (also known as the CapZβ-CapZα heterodimer), which capped the barbed ends of actin filaments and in this way prevented the further addition or loss of actin monomers, was a novel endocytic regulator. In further studies, I showed that CapZ docked to endocytic vesicles via its C-terminal actin-binding motif. Using ARPC1B proteins as the marker of branched actin networks, I found that CapZ knockout led to more actin branching around immature early endosomes (EEs); and this might impede the fusion between these vesicles, manifested by the small endocytic vesicles in CapZ-knockout cells. CapZ also recruits several RAB5 effectors, such as Rabaptin-5, to RAB5-positive early endosomes independent of its actin-binding domain, and this further activates RAB5 to promote early endosome maturation. Moreover, the conversion of PI(3)P to PI(3,5)P2 in the endosome might enable CapZ to be released from the matured early endosomes. In addition, the artificial tethering CapZ to early endosomes blocks endolysosomal trafficking. Finally, the depletion of CapZ results in immature endosomes, which prevents the endosomal escape of the Zika virus (ZIKV) RNA genome. Collectively, my results indicate that CapZ regulates endosomal trafficking by controlling the level of actin branching around early endosomes and recruiting RAB5 effectors.

In chapter four, I describe how, during the establishment of the CapZ interactome via a proximity-dependent Biotin Identification (BioID) technique, I identified a CapZ binding protein, called FAM129B, which was documented to be another novel regulator involved in the early-to-late endosome conversion process. My findings indicate that FAM129B might act as a RAB5 GAP to prevent RAB5 overaction. Moreover, FAM129B is also actively involved in the replacement of Rabex-5 by CCZ1 on the early endosome. The early endosomal recruitment of FAM129B depends on PI(3)P and active RAB5-GTP. However, when the K83 site of the FAM129B PH domain is mutated to alanine, the binding between RAB5 and FAM129B is dramatically reduced. In addition, the FAM129B-K83A mutant induces CCZ1 proteins to form an insoluble aggregate, which results in the loss of function of this protein. My results suggest that FAM129B regulates RAB5 activity by acting as its GAP and recruiting CCZ1, thereby promoting the early endosome to late endosome transition.