STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

Kana Aoki,Kenji Matsuzawa,Shota Harada,Keita Kawaji,Seiichi Uchida,Junichi Ikenouchi

doi:10.1038/s41467-020-20826-5

Kana Aoki, Kenji Matsuzawa + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-20826-5

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Journal: Nature Communications	Publication Date: Jan 20, 2021
Citations: 25	License type: open-access

Affiliation: Kyushu University

Abstract

The cytoplasm in mammalian cells is considered homogeneous. In this study, we report that the cytoplasmic fluidity is regulated in the blebbing cells; the cytoplasm of rapidly expanding membrane blebs is more disordered than the cytoplasm of retracting blebs. The increase of cytoplasmic fluidity in the expanding bleb is caused by a sharp rise in the calcium concentration. The STIM-Orai1 pathway regulates this rapid and restricted increase of calcium in the expanding blebs. Conversely, activated ERM protein binds to Orai1 to inhibit the store-operated calcium entry in retracting blebs, which results in decreased in cytoplasmic calcium, rapid reassembly of the actin cortex.

Highlights

Introduction of quantum dots into cellsQDs were introduced into DLD1 cells by electroporation using a NEPA21 Super Electroporator (NEPAGENE, Tokyo, Japan)
In order to visualize the fluidity of cytoplasm, fluorescent quantum dots (QDs) were introduced into DLD1 cells by electroporation to perform particle tracking analysis in the cytoplasm (Fig. 1c and Supplementary Movie 1)
The effects of convective flows derived from the influx of cytoplasm into the expanding blebs on the mobility of QDs was minimized by comparing the mobility of QDs at the late stage of expanding blebs and at the beginning of retracting blebs

Summary

Introduction

QDs were introduced into DLD1 cells by electroporation using a NEPA21 Super Electroporator (NEPAGENE, Tokyo, Japan). 1 μl QDs in 1 μl suspension buffer (Qtracker 605 Cell Labeling Kits (Invitrogen)) were suspended in 200 μl Opti-MEM (Invitrogen) and mixed with 1 × 106 DLD1 cells in a green cuvette with a 1-mm gap (NEPAGENE) and set to Electroporator. Poring pulse was optimized between 125 V and 275 V for 2.5 or 5.0 msec pulse length twice with a 50 msec interval between the pulses and 10% decay rate with + polarity. Cells were imaged after 1 h incubation by using a ×100 oil-immersion objective on an inverted microscope (IX83; Olympus corporation) interfaced to a spinning-disk confocal microscopy (Dragonfly[200]; OXFORD Instruments) equipped with a heating stage set to 37 °C. Images were captured every 20 msec (50 Hz) on a device camera and acquired using Fusion software (Dragonfly[200]; OXFORD Instruments)

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