1. Peri‐nuclear Rab34‐positive membranes contact lysosomes
Peri‐nuclear Rab34‐positive membranes contact lysosomes Representative confocal fluorescence image (left) showing methanol‐fixed HeLa cell transfected with GFP‐Rab34 and stained for LAMP1. Linescan analysis shows relative intensity of GFP and LAMP1 signals. Scale bar, 10 μm.Maximum intensity projection images from a confocal Z‐stack showing LAMP1 and endogenous Rab34 in P‐M‐fixed HeLa cells from normal and starved conditions. Whole cells and fields from which these panels are derived are shown in Fig EV4A. Boxes highlight regions shown in (C). Scale bar, 2 μm.Single confocal sections from the Z‐stack in (B). Orange arrows show close LAMP1/Rab34 association.Analysis of Lysotracker‐Red dynamics using PCC decay in HeLa cells transfected with either GFP or GFP‐Rab34. Data are from 10 cells per condition. Error bars show ± SEM. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Live‐cell spinning disc confocal image series at 10‐s intervals (from Movie EV3) showing GFP‐Rab34‐positive compartment making dynamic contacts with lysosomes. Scale bar, 1 μm.Live‐cell spinning disc confocal image series at 10‐s intervals (from Movie EV4) of FLCN‐GFP and mCherry‐Rab34 in the peri‐nuclear region of a FLCN‐GFP/HA‐FNIP2/mCherry‐Rab34 transfected HeLa cell. Blue arrow highlights FLCN–Rab34 association moving on a linear trajectory. Yellow arrow highlights shorter saltatory movements. White arrow highlights dynamic associations between distinct FLCN‐GFP and Rab34‐positive structures. Scale bar, 1 μm.Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells. Scale bar, 10 μm.
Georgina P Starling et al. EMBO Rep. 2016;17:
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