Optogenetic manipulation of cellular communication in axolotls


 Cells communicate through long cellular protrusions such as filopodia and neurites. However current approaches to study these contact-based cellular communication are largely limited to actin-depolymerizing drugs or genetic knockout of key actin modifiers which can cause severe cellular stress or semi-lethality in organisms. Here we present a versatile optogenetic toolbox of artificial myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo using light. Importantly, we discover that these long filopodial extensions are also gradually developed during axolotl limb regeneration, where we applied our toolbox to manipulate the composition and dynamics of these cellular extensions.


Introduction
Cells grow long cellular extensions to achieve highly e cient and accurate communication at a distance. Speci cally, prominent examples include specialized lopodia that tra c signaling proteins over long ranges during tissue development 1,2 , ne nger-like macrophage protrusion that are thought to be involved in pathogen clearance 3 , and invadopodia that integrate signals from the tumor microenvironment to facilitate tumor cell dissemination 4 . However we critically lack a genetically encoded tool that can selectively manipulate cellular protrusions with high spatiotemporal precision without affecting the global cytoskeletal network. In this work, we engineered a suite of such arti cial motors which we term "Arti cial Transport Vehicles" (ATVs), which are fast, highly processive, optogenetically controlled, and suitable for transporting GFP-tagged cargo at long ranges. Importantly, a major advantage of ATVs is the ability to speci cally manipulate cellular protrusions within dense cellular networks in living organisms in vivo. Regeneration is a process that has been reported to mimic normal embryonic development 5 , however the importance of lopodial extensions has not yet been investigated in this context. In this work, we describe live confocal imaging on the axolotl, a classic model system for tissue regeneration, at high spatial resolution in vivo, to investigate the role of lopodia in limb patterning during regeneration using our engineered myosin motors. Notably, we are able to directly manipulate the outgrowth of lopodial extensions in vivo, leading to the nding of an unexpected role of lopodial extensions in the context of cell signaling and tissue regeneration in axolotls. to be tagged by GFP (or any other variant that can be bound by GBP) at either the N-or C-terminus.

Reagents
Include the Piggybac transposon for stable integration into the genome to suit long-term activation.
2. Dilute the DNA constructs in DPBS to reach a nal concentration of 1-2 mg/ml. We recommend to test different molar ratios of the motor:cargo plasmids at the beginning. Start around 1:1. The optimal ratio may vary with different constructs. 6. Stretch out and position the forelimbs with sterile tweezers to enable injection and the subsequent electroporation.
7. Pressure-inject the DNA injection solution into the axolotl forelimb, approximately 1-2 mm distal to the elbow joint. Use 2-3 different injection sites to make sure the solution is fully diffused to different parts of the limb.
We recommend using multiple small pulses to inject the solution so that the site of injection can be more carefully controlled. 10. For optogenetic experiments, perform the anesthesia and mounting strictly in the dark. We used a red ashlight to aid the process. Use short pulses of 488 nm laser to activate optoATVs.

Troubleshooting
If not enough DNA injection solution can be injected into the forelimb: Page 7/8 -It is most likely because the tip of the glass needle is either too dull to penetrate the tissue or too sharp that it broke. Try to optimize the sharpness of the needle tip. Besides, also make sure the microinjector has enough force to inject the solution inside the forelimb e ciently.
If very limited uorescence can be detected from confocal imaging: -It may be caused by a failed electroporation. Please make sure the majority of the injection solution is inside the axolotl forelimb, determined by the presence of phenol red. Electroporation is typically strong enough with the parameters provided but can be further optimized. Check the quality of the plasmid solution and troubleshoot the microscope if electroporation is successful.
If optoATVs cannot be e ciently activated: -We have never experienced this problem if optoATVs were successfully expressed. It may be caused by mutations in the Cry2olig domain, and should be determined by sequencing.
If optoATVs are activated before light activation: OptoATVs are extremely sensitive, and should be very carefully protected from any potential light source. Make sure to conduct every animal procedure in strict dark. If strict dark condition cannot be guaranteed prior to imaging, we suggest waiting at dark for 30-60 minutes in the microscope prior to imaging for optoATVs to deactivate.

Time Taken
It takes approximately an hour for DNA construct preparation and axolotl electroporation.
After that wait for at least 3 days before performing confocal microscopy, which takes approximately an hour.

Anticipated Results
After e cient injection and electroporation, the majority of the forelimb is expected to be visibly red due to the presence of phenol red.