Functional and Mechanistic Studies of FAM129B Signaling in Endosomal Trafficking

Project: Research

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Description

Endosomal trafficking plays a vital role in various physiological processes, including migration, proliferation, immunity, and neurotransmission. The activation of RAB5 (a small GTPase) is essential for early endosome maturation, whereas its inactication is required for the early-tolate endosome transition. We previously illustrated a dual-functional role of CapZ (a canonical actin capping protein) in endosomal trafficking: it controls actin filament density around immature early endosomes via its C-terminal domain to facilitate the homotypic fusion of the endocytic vesicles; and it functions as a scaffold protein via its N-terminal domain to recruitRAB5 effectors to early endosomes, thereby establishing a positive feedback loop to induce early endosome maturation. Particularly, we showed that CapZ knockout induced the accumulation of smaller early endosomes, and significantly inhibited the endosomal trafficking of transferrin and integrins when compared to the control cells. CapZ knockout also significantly inhibited cell migration in vitro and the metastasis of breast cancer cells in an orthotopic cancer mouse model. To further understand the role of CapZ in endosomal trafficking and metastasis, we established a protein interaction network of CapZ by performing BioID proximity labeling and affinity purification. Our current study identified FAM129B, a PH domain-containing protein, as one of the CapZ’s binding proteins. We showed that FAM129B interacted with CapZ, RAB5, and several RAB5 effectors or regulators (e.g., Rabex-5, CCZ1-Mon1, and VPS34) by Co-IP experiments. FAM129B was closely associated with early endosomes. In contrast to CapZ-knockout cells, in FAM129B-knockout cells established by CRISPR/Cas9, the size of the early endosomes was larger and the number of early endosomes was smaller when compared to the control cells, and addback of FAM129B to FAM129B-knockout HeLa cells restored the size and number of early endosomes to those in control cells. FAM129B knockout also increased RAB5-GTP levels. Moreover, FAM129B knockout compromised endosomal trafficking of EGFR, transferrin, and integrins, and significantly inhibited migration and single colony formation of cancer cells. We hypothesize that the interplay between FAM129B and CapZ or other RAB5 effectors or regulators participates in RAB5 inactivation during the early-to-late endosome transition. Therefore, here we propose to: (1) determine the interplay between FAM129B and CapZ in endosomal trafficking; and (2) further dissect the molecular mechanisms underlying FAM129B-mediated RAB5 regulation. Given that dysfunctional endosomal trafficking has been associated with many human diseases, e.g., cancer, the success of this project will shed light on the mechanisms underlying endosomal trafficking, and reveal novel drug targets for therapy against human diseases.

Detail(s)

Project number9043416
Grant typeGRF
StatusActive
Effective start/end date1/07/22 → …