Ca2+/calmodulin-dependent protein kinase II β (CaMKIIβ) decodes ER Ca2+ transients to trigger autophagosome formation. Qiaoxia Zheng et al.

Description

These are unprocessed and uncompressed imaging data (microscopy as well as gels and blots) of the study "Ca2+/calmodulin-dependent protein kinase II β (CaMKIIβ) decodes ER Ca2+ transients to trigger autophagosome formation". In this study, we focused on how Ca2+ transients on the ER outer surface elicited by autophagy stimuli are sustained and decoded to trigger autophagosome formation in multicellular organisms. Here we show that CaMKIIβ integrates ER Ca2+ transients to trigger liquid-liquid phase separation (LLPS) of the autophagosome-initiating FIP200 complex. In response to ER Ca2+ transients, CaMKIIβ is recruited from actin filaments and forms condensates, which serve as sites for emergence of or interaction with FIP200 puncta. CaMKIIβ phosphorylates FIP200 at Thr269, Thr1127 and Ser1484 to modulate LLPS and properties of the FIP200 complex, thereby controlling its function in autophagosome formation. CaMKIIβ also controls the amplitude, duration and propagation of ER Ca2+ transients during autophagy induction. CaMKIIβ mutations identified in the neurodevelopmental disorder MRD54 affect the function of CaMKIIβ in autophagy. Our study reveals that CaMKIIβ is essential for sustaining and decoding ER Ca2+ transients to specify autophagosome formation in metazoans.

Steps to reproduce

In this study, we showed lots of images of Multi-modal SIM (multi-SIM) super-resolution living-cell imaging. The multi-SIM system integrates TIRF-SIM, grazing incidence (GI-SIM) (Guo et al., 2018) and 3D-SIM, as well as lattice light sheet microscopy (LLSM). Nonlinear SIM achieves an optical resolution of 60.8 nm at a frame rate of more than 20 Hz. GI-SIM achieves an imaging speed of up to 684 Hz for 60,000 frames. This multi-modal SIM system allows us to select the optimum imaging mode for a specific process according to its subcellular location, dynamics, duration, etc. The NA of the multi-SIM lens is 1.49 (Nikon CFI SR HP Apo TIRF 100×/1.49 Oil objective lens). For multi-SIM imaging experiments, COS7 cells transfected with the indicated plasmids or infected by lentivirus were seeded onto Mattek glass-bottomed dishes 18-24 hours prior to imaging. Cells were maintained in 37 °C and 5% CO2 during imaging. Multi-SIM images were analyzed by Image J.

Institutions

Categories

Funding

Licence