How to apply FLUCS in single cells and living embryos

Abstract

This protocol is associated to the publication "Non-invasive perturbations of intracellular flow reveal physical principles of cell organization" by Mittasch et al., published in Nature Cell Biology. In the following, we describe the procedure for maintenance and preparation of C. elegans zygotes for imaging and application of FLUCS. Preparation of S. cerevisiae cells for microscopy and intracellular flow perturbations. S. cerevisiae cells were grown into logarithmic stage. Subsequently the S. cerevisiae cells were either grown for 2 hours or alternatively energy depleted through addition of 2-Desoxy-D-Glucose (inhibition of glycolysis) and antimycin A (inhibition of mitochondrial ATP production) for 2 h prior to flow induction. This treatment causes a more than 95% reduction in cellular ATP 2. Potentially this protocol is suitable to energy deplete all S. cerevisiae strains and closely related yeast species. A specific marker such as µNS-GFP1is needed to observe subsequent flow perturbations by microscopy.

Fungal Wound Healing through Instantaneous Protoplasmic Gelation

“…This work adds protoplasmic gelation to biological processes where variation in flow appears to play a deterministic regulatory role. 38 At the tissue level, these include blood clotting in vertebrates 13 and silk spinning in spiders, 39 and in cells, symmetry breaking through cortical flow in zygotes 40,41 and the formation of distinct sub-compartments through regimented cytoplasmic streaming in fungal hyphae. 6 Like protoplasmic gelation defined here, silk-spinning is irreversible and has a basis in the formation of b-sheets.…”

Active gelation breaks time-reversal-symmetry of mitotic chromosome mechanics

“…Herein, we built on recent methodological advancement that allows us to optically induce hydrodynamic flows within living cells and developing embryos 5,6 , a technique we refer to as focused-light-induced cytoplasmic streaming (FLUCS). Physically, FLUCS makes use of thermoviscous flows 7 , which have been also used to distinguish between fluid and gel like states of the cytoplasm in yeast cells 5 .…”