Live cell imaging of protein trafficking: Dynamics and functional insights

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal-multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalizes with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Super-resolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knock-down, cells in mouse cortical neurons reduce their surface GluA1 level. These results suggest that, unlike the Human Immunodeficiency Virus Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), Arc capsids assemble at the endocytic membranes is mediated by PI3P. Understanding Arc’s secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Mehta, Kritika

Contributors dc:contributor

• Zhang, Kai
• Fratti, Rutillo A
• Tsai, Nien-Pe
• Stadtmueller, Beth M

• Activity-regulated Cytoskeleton-associated Protein
• Phospholipids
• Virus-like Capsid
• Fluorescence Imaging
• Super Resolution Imaging
• Minflux

Statement dc:rights

• © 2024 Kritika Mehta

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/125773

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F125773