Semi-Intact Cell System for Reconstituting and Analyzing Cellular Golgi Dynamics

Microscopic image-based covariation network analysis for actin scaffold-modified insulin signaling

“…We have previously shown frequent, prominent accumulation of round-shaped structures of actin fibers in hepatoma-derived H4IIEC3 cells upon insulin stimulation (Kano and Murata, 2019). Here, live imaging using H4IIEC3 cells transiently expressing LifeAct-EGFP (Riedl et al, 2008) revealed, upon insulin stimulation, the rearrangement of actin filaments and subsequent actin waves just beneath the plasma membrane (Figure 1A,Video S1).…”

“…We suppose that the concentration of activated Akt (pSer473), PI(3,4)P 2 , and actin-binding proteins such as SNX9 and Arp2/3 by a similar mechanism may induce the association of downstream insulin-signaling molecules, such as GSK3b (pSer9), mTOR, and mTOR (pS2344), to the actin scaffold, thereby initiating spatial-dependent regulation of glycogenesis or insulin-stimulated protein synthesis. Interestingly, PI3P on early endosomes may also be involved in this process because PI3P functions in recruiting actin-nucleating proteins, such as SNX9 (Daste et al, 2017) and WASH (Singla et al, 2019), and we also previously showed a role of PI3P on actin scaffold formation (Kano and Murata, 2019).…”

“…Using high-resolution microscopy, we found actin mesh-like structures of the actin scaffold with Akt (pSer473) aligned on the actin fibers (Figure S1A). In a previous study (Kano and Murata, 2019), accumulation to the actin scaffold of actin-modulating proteins, such as sorting nexin 9 (SNX9) and actin-related protein 2 and 3 (Arp2/3), and endosomal proteins, such as early endosome antigen 1 (EEA1) and adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1), were also observed. These findings suggest that the actin scaffold is a transient membrane-associated structure, likely derived from multiple early endosomes surrounded by an actin mesh, created to accommodate the transient accumulation of a variety of signaling proteins to modulate insulin signal transduction.…”

“…The plasmid encoding LifeAct-EGFP was produced as described previously (Kano and Murata, 2019). H4IIEC3 cells were grown on glass-bottomed dishes (Iwaki, 4970-011) coated with collagen Type-IC.…”

Development of a Pseudocellular System to Quantify Specific Interactions Determining the G-Quadruplex Function in Cells

“…The resealing semi-intact cell technique was previously reported, involving the removal of intracellular fluid from diseased cells and its transfer to normal cells to generate model cells. , This technique proves to be valuable in comprehending the effects of intracellular fluids from diseased cells on target molecules. In addition, in-cell NMR is also a useful method for understanding the behavior of nucleic acids in cells .…”