Malchenko, N. | Search bioRxiv

bioRxiv2025

Fluorescence lifetime-based biosensor for monitoring compartmentalized autophagy dynamics in the intact mammalian brain

Autophagy is a key process in regulation of neuronal development, plasticity, and local metabolism. Yet, autophagy dynamics and regulation within intact neuronal circuits remains poorly understood. Here, we developed a pH sensitive fluorescence lifetime-based imaging method which allows to monitor autophagy dynamics in the living mouse brain. This approach allowed us to uncover compartmentalized autophagic activity across soma, dendrites, and axons of layer 2/3 cortical neurons. We found pronounced differences in dendritic dynamics of autophagic vesicles in vivo, where distal dendrites showed elevated vesicle motility compared to proximal dendrites. Notably, sensory experience modulated dendritic autophagy dynamics in the somatosensory cortex. We further combined in vivo autophagy imaging with disease associated genetic perturbation to uncover novel autophagy related phenotypes. Altogether, this approach highlights the spatial and functional complexity of autophagy in the intact mammalian brain and establishes a framework for investigating its role in synaptic regulation during development, plasticity, and aging.

neuroscience↗

bioRxiv2024

Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain

The phosphatase and tensin homolog (PTEN) is a vital signaling protein which maintains an inhibitory brake that is critical for cellular metabolism, proliferation, and growth. The importance of PTEN signaling is evident from the broad spectrum of human pathologies associated with its loss of function. Moreover, loss or gain of PTEN function in animal models leads to aberrant cellular morphology, function, and metabolic regulation. However, despite the important role of PTEN signaling, there is currently no method to dynamically monitor its activity with cellular specificity within intact biological systems. Here, we describe the development of a novel PTEN biosensor, optimized for two-photon fluorescence lifetime imaging microscopy (2pFLIM). This biosensor is designed to measure PTEN activity within intact cells, tissues, and organisms. Our approach is based on monitoring FRET-dependent changes in PTEN conformation, which serves as a proxy for the activity state in living cells. We identify a point mutation that allow us to express this biosensor with minimal interference to endogenous PTEN signaling and cellular function. We demonstrate the utility of imaging PTEN signaling in cell lines, developing C. elegans, and in the living mouse brain. To complement this approach, we developed a red-shifted PTEN sensor variant that permits simultaneous imaging with GFP-based sensors. Finally, we use in vivo PTEN imaging in the mouse brain to identify cell-type specific dynamics of PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.

neuroscience↗