Date: Thu July 3rd 2025
Time: 3pm UK time (other times)
Place: Zoom link
Previous Meeting: issue #26
Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Dr Eve Carter (UCL), Prof Matt Todd (UCL), Prof Karl Swann (Uni Cardiff), Prof Nicola Burgess-Brown (UCL), Prof Opher Gileadi (SGC), Anna Girtle (Uni Toronto).
Decks:
Agenda:
In meeting:
Karl presented recent functional assay data on the Nuvisan compound, focusing on experiments with mouse eggs injected with PLCz1 mRNA or protein to monitor calcium oscillations. Initial tests at 1 μM showed no effect, while 5 μM produced partial inhibition after extended incubation and 10 μM blocked sperm-egg fusion entirely. However, PLCz1‑induced oscillations themselves were not blocked. In fact, they continued to persist at 10 μM and only stopped at 50 μM, which is a toxic concentration that caused blebbing. Based on these findings, Karl suggested the compound is likely acting at the level of sperm-egg fusion rather than directly inhibiting PLCz1 activity.
Matthew and Opher asked whether catalytically inactive PLC constructs or other PLC isoforms had been tested, which Karl confirmed. Karl's prior studies showed only PLCz1 reliably induces oscillations (catalytically inactive PLCz1 results in no oscillations), with other isoforms requiring unnaturally high doses. They also discussed possible next experiments to clarify the mechanism such as microinjecting purified PLCz1 protein pre‑mixed with the compound to bypass membrane‑permeation issues and using chicken PLCz1 protein. Lastly, possible controls were discussed such as injecting DMSO alone to account for solvent effects, and comparing results to propranolol, a known but non-specific oscillation inhibitor that acts indirectly by depleting PIP2.
Karl introduced some other potential issues, such as whether the compound partitions into egg membranes (potentially explaining fusion inhibition) and solubility limitations (maximum workable concentrations in DMSO). He noted that other lipophilic molecules and fluorescent dyes have shown similar fusion‑blocking behavior, so this mechanism of action is not unheard of. Ultimately, while the compound does inhibit fertilization, its failure to suppress PLCz1 oscillations makes it unsuitable for probing PLCz1 function.
To do:
L'esprit de l'escalier
If you'd like to follow up after the meeting, please comment below. You can also email, but please be clear if anything in the email should not be public domain - the default is open.
Next meeting: Tue August 12th 2025, 3pm UK time
Date: Thu July 3rd 2025
Time: 3pm UK time (other times)
Place: Zoom link
Previous Meeting: issue #26
Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Dr Eve Carter (UCL), Prof Matt Todd (UCL), Prof Karl Swann (Uni Cardiff), Prof Nicola Burgess-Brown (UCL), Prof Opher Gileadi (SGC), Anna Girtle (Uni Toronto).
Decks:
Agenda:
In meeting:
Karl presented recent functional assay data on the Nuvisan compound, focusing on experiments with mouse eggs injected with PLCz1 mRNA or protein to monitor calcium oscillations. Initial tests at 1 μM showed no effect, while 5 μM produced partial inhibition after extended incubation and 10 μM blocked sperm-egg fusion entirely. However, PLCz1‑induced oscillations themselves were not blocked. In fact, they continued to persist at 10 μM and only stopped at 50 μM, which is a toxic concentration that caused blebbing. Based on these findings, Karl suggested the compound is likely acting at the level of sperm-egg fusion rather than directly inhibiting PLCz1 activity.
Matthew and Opher asked whether catalytically inactive PLC constructs or other PLC isoforms had been tested, which Karl confirmed. Karl's prior studies showed only PLCz1 reliably induces oscillations (catalytically inactive PLCz1 results in no oscillations), with other isoforms requiring unnaturally high doses. They also discussed possible next experiments to clarify the mechanism such as microinjecting purified PLCz1 protein pre‑mixed with the compound to bypass membrane‑permeation issues and using chicken PLCz1 protein. Lastly, possible controls were discussed such as injecting DMSO alone to account for solvent effects, and comparing results to propranolol, a known but non-specific oscillation inhibitor that acts indirectly by depleting PIP2.
Karl introduced some other potential issues, such as whether the compound partitions into egg membranes (potentially explaining fusion inhibition) and solubility limitations (maximum workable concentrations in DMSO). He noted that other lipophilic molecules and fluorescent dyes have shown similar fusion‑blocking behavior, so this mechanism of action is not unheard of. Ultimately, while the compound does inhibit fertilization, its failure to suppress PLCz1 oscillations makes it unsuitable for probing PLCz1 function.
To do:
L'esprit de l'escalier
If you'd like to follow up after the meeting, please comment below. You can also email, but please be clear if anything in the email should not be public domain - the default is open.
Next meeting: Tue August 12th 2025, 3pm UK time