Characterization of Beclin 1-Actin Interaction and the Impact of Beclin 1-PIST in Leishmania major Infection

Gadpayle, Mandip Pratham (2024) Characterization of Beclin 1-Actin Interaction and the Impact of Beclin 1-PIST in Leishmania major Infection. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Mandip Pratham Gadpayle (16RS015)) 16RS015.pdf - Submitted Version Restricted to Repository staff only Download (6MB)

Abstract

The actin cytoskeleton plays a crucial role in governing essential cellular functions, including autophagy, a highly conserved process vital for maintaining cellular homeostasis. While numerous ATG proteins are known to regulate autophagy, only a handful of actin-binding proteins have been identified in the autophagy-actin-cytoskeleton connection. Beclin 1, a pivotal regulator of autophagy, orchestrates this process by forming complexes with ATG14, UVRAG, or PIST, alongside Class III PI3KC3. Notably, the Beclin 1-PIST complex has been implicated in non-canonical autophagy during infection, facilitating pathogen clearance. However, the precise role of actin in early autophagic events remains poorly understood. Furthermore, the involvement of the Beclin 1-PIST complex in intracellular parasitic infections remains unexplored. In this study, we uncover the interaction between Beclin 1 and actin and explore the functional significance of the Beclin 1-PIST complex during Leishmania major infection. Our investigation reveals that Beclin 1 possesses putative WH2-like domains in its coiled-coil region, with in-vitro actin-interaction studies confirming the interaction of Beclin 1’s coiled-coil domain with actin. Moreover, we demonstrate that Beclin 1 sequesters actin monomers and promotes actin filament depolymerization. Additionally, we demonstrate the colocalization of Beclin 1 puncta with phalloidin labelled-actin filaments in HeLa cells. Furthermore, we observe the recruitment of the Beclin 1-PIST complex to L. major-containing compartments. Upon L. major infection, PIST translocates from the trans-Golgi network to the vicinity of the parasite’s nucleus in an infection-specific manner. Although the expression level of PIST remains unchanged during infection, its distribution undergoes alteration. Genetic manipulation of PIST results in the modulation of parasite burden in macrophages. Additionally, we find that the recruitment of LC3 to L. major-harboring vacuoles is inhibited. In summary, our findings highlight the direct interaction between Beclin 1 and actin and underscore the regulatory role of the Beclin 1-PIST in governing Leishmania major infection dynamics.

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