Tal Laviv

Thanks so much Mike!

Thanks so much!

We are further perusing shing this approach in many different directions- different cell types in the brain, and completely different model systems to monitor autophagy and pH using this general approach so hope much more to come in the future.

All of this would not be possible without the support and funding from the Chan Zuckerberg Initiative that contributed to this work at a critical step and really allowed us to pursue grand challenges.

This work was possible due to critical contribution from Ben Scholl that helped us analyze vesicle movement in vivo, which was not an easy challenge. Brock Grill and Muriel Dubois were key driving forces to implement this sensor in C elegans

Much more in the paper! Including some evidence that dendritic autophagy dynamics is experience dependent! This work was a true team effort led by brilliant student Maya Maman, with critical help from lab members.

The most striking feature is that the sensitivity of this approach allows to track autophagic vesicle transport in vivo for the first time! We found that distal dendritic segments showed elevated speed of autophagic vesicles, while proximal dendrites were more stable.

Using 2pFLIM, we were able to gain access to autophagic levels in cell bodies, dendrites and axons. One of the striking things we found was that autophagy levels are vastly different across different dendritic compartments, which were previously unknown.

We benchmarked this approach in a number of models, cell lines, neuronal cultures and even C. elegans. Most importantly, this robust approach now allows us to monitor autophagy levels in the living mouse brain!

We used fluorescence lifetime (FLIM) to solve this problem! By engineering a new FRET pair, we standardize pH-based monitoring in cells. We used a recently discovered pH insensitive FRET donor, named TOLLES, which showed high resilience and its lifetime was pH independent.

This approach has been instrumental studying autophagy in neuronal cultures or small organisms. However, monitoring autophagy in mammalian brains remains challenging, since it is difficult to detect small changes in fluorescence intensity, in particular in acidic compartments.

This approach allows to use the properties of GFP and RFP to determine the state of autophagy- since GFP is easily degraded in acidic compartments, the transition from autophagosomes to autolysosome is associated with GFP degradation.

While numerus approaches were developed to measure autophagy, tracking autophagy in living cells relied primarily on fluorescent intensity. The principle of this approach relies on tagging LC3, an essential component of autophagy vesicles with a GFP-RFP fusion.

We know that autophagy is critical for normal brain development- its disruption in early stages leads to brain dysfunction and neurodegeneration. Autophagy dysfunction is indeed associated with many human pathologies- in development and also during neurodegeneration.

Autophagy is a central mechanism which allows cells to maintain homeostasis, by allowing efficient degradation of proteins, lipids and organelles. Generally, autophagy is triggered in response to various stress signals, which indicate reduction in energy levels.

Fluorescence lifetime-based biosensor for monitoring compartmentalized autophagy dynamics in the intact mammalian brain Synaptic architecture underlies neuronal computation, with autophagy serving as a key regulator of synaptic plasticity, function and local metabolism. Yet, autophagy dynamics and regulation within int...

I am excited to share our new paper, where we developed and used a new approach that allows us to dynamically monitor autophagy in the intact mouse brain! biorxiv.org/cgi/content/...

Thanks Kasia!

Thanks Vidhya!

Thanks Hyungbae!

Thanks!

Thanks so much Tommaso!

Thanks!

Thanks Nicolas!

Thanks so much!!

Thank you!

Thanks Rita!

Thanks Jonas!!

Thanks Onur!

This project was spearheaded by a Tomar Kagan, a talented PhD student in my lab. special thanks for @ritastrack.bsky.social and nature methods team for their professional and supportive process to improve the paper to its present form, it was a real pleasure to work with them.

This highlights the need to explore PTEN signaling in a cell-specific manner, and to further explore the original findings and associate PTEN signaling during early brain development! I would like to thank all the collaborators that helped us in this project, and my lab members for their hard work.