Mneongreen

When Atto 532 (A532, Sigma) was included in the bath solution, the dye concentration was 30 μM. The PH-EGFP (phospholipase C delta PH domain attached with EGFP) was obtained from Dr. Tamas Balla. PH-mNeonGreen (PH_G) and PH-mTFP1 construct were created by replacing the EGFP tag of PH-EGFP with mNeonGreen (Allele Biotechnology)^{51 (link)} or mTFP1 (Addgene), respectively. Dynamin 1-K44A-mRFP was purchased from Addgene (#55795). Dynamin 2-mTFP1 (or dynamin 1-mTFP1) and dynamin 2-mNeonGreen construct were created by replacing the EGFP tag of dynamin 2-EGFP (or dynamin 1-EGFP, Addgene) with mTFP1 or mNeoGreen, respectively. Lifeact-mTFP1 construct was created by replacing the TagGFP2 of lifeact-TagGFP2 (Ibidi) with mTFP1.

We constructed all sensors by polymerase chain reaction (PCR) cloning. The template for the mScarlet moiety of the protein fusions originated from pmScarlet_C1 (#85042, Addgene). The template for the Troponin C (TnC) moieties originated from Twitch-2B (#100040, Addgene) and Twitch-3 (#49532, Addgene). The sfGFP, mNeon, mCitrine, mVenus, and mClover moieties of the fused proteins originated from sequences of msfGFP (# 91902, Addgene), mNeonGreen (#58179, Addgene), Twitch-3 (#49532, Addgene), Twitch-2B (#100040, Addgene), and mClover3 (#74252, Addgene), respectively. We added a nuclear-export sequence (MLQNELALKLAGLDINKTG)^{10 (link),56 (link)} at the N-terminus to localize the sensor expression to the cytoplasm. The sequences of all sensors are in Supplementary Table 2. To express the fused proteins in HEK293 cells, we inserted the genes into a lentivirus backbone under the CamkIIα promoter (#48762, Addgene). To express the fused proteins in cultured neurons and in live mice, we inserted the genes into an adeno-associated virus backbone under the CamkIIα promoter (#26969, Addgene). To express the fused protein in bacteria, we inserted the genes into the pET-28b backbone (69865-3, Millipore Sigma) while fusing the sensors to a 6× His tag.