Skip to main content
Fig. 8 | BMC Biology

Fig. 8

From: A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

Fig. 8

Targeting CALI to lysosomes using RNb-SNAPf reduces lysosomal motility. a Schematic of RNb-SNAPf after labelling with SNAP-Cell-fluorescein (green circle) and bound to RFP. b HeLa cells co-expressing LAMP1-mCh and RNb-SNAPf were incubated with SNAP-Cell-fluorescein (0.5 μM, 30 min, 37 °C), which labelled lysosomes (colocalization values, r = 0.73 ± 0.02, n = 6 cells), and imaged using TIRFM. c, d Cells were then exposed to 488-nm light for 3 s to induce CALI. TIRFM images show a representative cell at different times before (c) and after (d) CALI, with the image at t = 0 s shown in magenta and the image at t = 60 s in green. White in the merged images from the two different times indicates immobile lysosomes, while green and magenta indicate lysosomes that moved in the interval between images. Yellow boxes show regions enlarged in subsequent images. Scale bars 10 μm (main images) and 2.5 μm (enlargements). For clarity, images were auto-adjusted for brightness and contrast (ImageJ) to compensate for bleaching of mCh during tracking and CALI. e Effect of CALI on the displacements of individual lysosomes, determining by particle-tracking (TrackMate), during a 60-s recording from a representative cell (images taken every 1 s; mean values shown by bars). f Summary data (mean ± SEM, n = 6 cells from 6 independent experiments) show the mean fractional decrease in displacement (Δ displacement) due to CALI in cells expressing RNb-SNAPf or cytosolic SNAPf (see Additional file 1: Figure S2). The fractional decrease in displacement for each cell was defined as (MDpre–MDpost)/MDpre, where MDpre and MDpost are the mean displacement of all tracked particles in 60 s before and after CALI. *P < 0.05, unpaired Student’s t test

Back to article page