Fig. 6: Peripheral lysosomes associate with small GTPase Rac1 and regulates its signaling at sites of growing lamellipodia. | Nature Communications

Fig. 6: Peripheral lysosomes associate with small GTPase Rac1 and regulates its signaling at sites of growing lamellipodia.

From: Mechanosensitive dynamics of lysosomes along microtubules regulate leader cell emergence during collective cell migration

Fig. 6: Peripheral lysosomes associate with small GTPase Rac1 and regulates its signaling at sites of growing lamellipodia.The alternative text for this image may have been generated using AI.

A MDCK cells allowed to migrate for T-2 h were PFA fixed, immunostained for endogenous RAC1 and lysosomes (Anti-LAMP1), and imaged on an Airy-Scan super resolution microscope. Yellow arrowhead marks endogenous RAC1 co-localized with LAMP1. B Line plot showing the overlap of endogenous RAC1 and lysosomes. C Representative confocal micrograph of mouse embryonic skin wound allowed to migrate for 30 min, PFA fixed and immunostained for endogenous RAC1, lysosomes (Anti-LAMTOR-4) and Actin (Alexa-fluor conjugated phalloidin). Arrowheads mark colocalized RAC1 and LAMTOR-4. D The line plot shows the overlap between endogenous RAC1 and lysosomes in mouse embryonic skin wounds. E Representative immunoblot of organelle fractionated from MDCK cells on an optiprep gradient and probed for lysosomes (Anti-LAMP1), RAC1 (Anti-Rac1), mitochondria (Anti-Tom20), and early endosomes (Anti-EEA1). F1, F3 and F4 are exclusive lysosome fractions and are positive for endogenous RAC1. F Live imaging snapshots of MDCK cells expressing GFP-RAC1WT and Dextran-647 labeled lysosomes. The cells were allowed to migrate for 2 h, followed by live imaging of a cell at the migrating edge on a confocal microscope. The yellow arrowheads mark co-localized GFP-RAC1WT and Dextran-647 labeled lysosomes. G Schematic depicting the expression of mEOS-Rac1 and photoconversion using UV light, leading to a green to red shift in emission, thus allowing tracking of its localization dynamics. H MDCK cells transiently transfected with mEos-Rac1 and dextran-647 labeled lysosomes were allowed to migrate for 60 min before photoconversion to initiate lamellipodia formation. Dynamics of Rac1 puncta formation and trajectory were followed for photoconverted mEos-Rac1. Yellow arrowheads mark emerging Rac1 puncta and their localization to dextran-647 labeled lysosomes. Kymograph representing the colocalization dynamics of lysosomes and photoconverted mEos-Rac1. I–K Representative heat maps of FRET-based Rac1 activity sensor for control, RAC1 inhibitor (NSC), and kinesore treated MDCK cells. L Mean FRET index was calculated for control and kinesore treated cells at T-0 and T-2 h and represented as a scatter-column graph (from left to right n = 59, 47, 44 and 46 from 3 independent experiments). Statistical analysis was performed using the one-way ANOVA test. Scale bar-10 µm; *, ** and **** signify p-value < 0.05, 0.01 and 0.0001, respectively. All data are mean ± s.e.m.

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