TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role 
in mitophagy, the selective clearance of damaged mitochondria. More than 90 
distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and 
fronto-temporal dementia, including missense mutations that disrupt the 
abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin 
(OPTN), autoactivate, or catalyze phosphorylation. We investigated how 
ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging 
to dissect the molecular mechanisms involved in clearing damaged mitochondria. 
Some mutations cause severe dysregulation of the pathway, while others induce 
limited disruption. Mutations that abolish either TBK1 dimerization or kinase 
activity were insufficient to fully inhibit mitophagy, while mutations that 
reduced both dimerization and kinase activity were more disruptive. Ultimately, 
both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for 
engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we 
find that ULK1 activity contributes to the phosphorylation of OPTN in the 
presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 
mutants induce mitochondrial stress under basal conditions; network stress is 
exacerbated with further mitochondrial insult. Our study further refines the 
model for TBK1 function in mitophagy, demonstrating that some ALS-linked 
mutations likely contribute to disease pathogenesis by inducing mitochondrial 
stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less 
impact on mitophagy in our assays, suggesting that cell-type-specific effects, 
cumulative damage, or alternative TBK1-dependent pathways such as innate 
immunity and inflammation also factor into the development of ALS in affected 
individuals.