Biology subjects

Padman, B. S.

Publications and source records attributed to Padman, B. S..

4 recordsLinked to original sources

bioRxiv2024

AI-directed voxel extraction and volume EM identify intrusions as sites of mitochondrial contact

Membrane contact sites (MCS) establish organelle interactomes in cells to enable communication and exchange of materials. Volume electron microscopy (vEM) is ideally suited for MCS analyses, but semantic segmentation of large vEM datasets remains challenging. Recent adoption of artificial intelligence (AI) for segmentation has greatly enhanced our analysis capabilities. However, we show that organelle boundaries, which are important for defining MCS, are the least confident predictions made by AI. We outline a segmentation strategy termed AI-directed Voxel Extraction (AIVE), that combines AI predictions with image electron signals to confidently segment membrane boundaries irrespective of the AI model used. We demonstrate the precision conferred by AIVE by applying it to the quantitative analysis of organelle interactomes from multiple FIB-SEM datasets. Through AIVE, we discover a previously unknown category of mitochondrial contact that we term the mitochondrial intrusion. We hypothesise that intrusions serve as anchors that stabilize MCS and promote organelle communication.

cell biology↗

bioRxiv2023

Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD

Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PINK1/Parkin-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 adaptors NAP1 and SINTBAD restrict the initiation of OPTN-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of NDP52-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.

cell biology↗

bioRxiv2022

Unconventional Initiation of PINK1/Parkin Mitophagy by Optineurin

Cargo sequestration is a fundamental step of selective autophagy in which cells generate a double membrane structure termed an autophagosome on the surface of cargoes. NDP52, TAX1BP1 and p62 bind FIP200 which recruits the ULK1/2 complex to initiate autophagosome formation on cargoes. How OPTN initiates autophagosome formation during selective autophagy remains unknown despite its importance in neurodegeneration. Here, we uncover an unconventional path of PINK1/Parkin mitophagy initiation by OPTN that does not begin with FIP200 binding nor require the ULK1/2 kinases. Using gene-edited cell lines and in vitro reconstitutions, we show that OPTN utilizes the kinase TBK1 which binds directly to the class III phosphatidylinositol 3-kinase complex I to initiate mitophagy. During NDP52 mitophagy initiation, TBK1 is functionally redundant with ULK1/2, classifying TBK1s role as a selective autophagy initiating kinase. Overall, this work reveals that OPTN mitophagy initiation is mechanistically distinct and highlights the mechanistic plasticity of selective autophagy pathways.

cell biology↗

bioRxiv2020

Atg4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The Atg4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like Atg8 family (LC3/GABARAPs). We make the unexpected discovery that human Atg4s can directly promote autophagosome formation independently of their protease activity and of Atg8 family processing. High resolution structures of phagophores generated with artificial intelligence-directed 3D electron microscopy reveal a role for the Atg4 family in promoting phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. Atg4 proximity interaction networks stimulated by PINK1/Parkin mitophagy are consistent with roles for Atg4s in protein/vesicle transport and lipid modification. We also show that Atg8 removal during autophagosome maturation does not depend on Atg4 de-lipidation activity as previously thought. Instead, we find that Atg4s can disassemble Atg8-protein conjugates, revealing a role for Atg4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the Atg4 family beyond the Atg8 lipidation axis and provide an AI driven framework for high-throughput 3D electron microscopy.

cell biology↗