Real-time single-molecule imaging of CaMKII-calmodulin interactions

Area of Science:

• Biochemistry and Molecular Biology
• Neuroscience
• Cell Signaling

Background:

• Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in cellular signaling pathways.
• CaMKII activation involves calcium/calmodulin (CAM) binding and ATP-dependent autophosphorylation, which enhances CAM affinity.
• Understanding the precise mechanisms of CAM association with CaMKII, particularly its ATP dependence, is vital for deciphering its regulatory functions.

Purpose of the Study:

• To investigate the ATP dependence of CAM association with the CaMKIIβ isoform using single-molecule imaging.
• To elucidate the role of CaMKII dimerization and specific phosphorylation sites in regulating CAM binding affinity.
• To compare the behavior of wild-type CaMKII with various mutants under different ATP conditions.

Main Methods:

• Single-molecule total internal reflection fluorescence microscopy (TIRFm) to observe Rhodamine-CAM association with Venus-CaMKIIβ.
• High-resolution (0.5 s) tracking of CAM binding and dissociation events.
• Spot-photobleaching to determine CaMKII holoenzyme stoichiometry simultaneously with CAM occupancy.
• Utilized phosphorylation-defective, ATP-binding impaired, and phosphomimetic CaMKII mutants.

Main Results:

• ATP-dependent CAM association with CaMKII requires dimer formation for both α and β isoforms.
• CaMKII mutants revealed distinct low- and high-affinity states for CAM binding, modulated by ATP and phosphorylation.
• Wild-type CaMKII exhibited a bimodal distribution of low- and high-affinity states in ATP, unlike previously published in vitro assays.
• The phosphomimetic mutant (T287D) showed significantly higher CAM affinity, while ATP-binding impaired mutants displayed reduced affinity.

Conclusions:

• CaMKII holoenzyme structure, dimerization, and phosphorylation status critically regulate CAM binding affinity in an ATP-dependent manner.
• The observed bimodal distribution in wild-type CaMKII suggests complex in vivo regulation of CAM association.
• Findings highlight how assay conditions can influence the perceived balance of CaMKII activation and inhibition, impacting kinase function in vivo.