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. 2016 Jun;17(6):823-41.
doi: 10.15252/embr.201541382. Epub 2016 Apr 13.

Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes

Georgina P Starling  1 , Yan Y Yip  1 , Anneri Sanger  1 , Penny E Morton  1 , Emily R Eden  2 , Mark P Dodding  3
Affiliations

Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes

Georgina P Starling et al. EMBO Rep. 2016 Jun.

Abstract

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt-Hoge-Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri-nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi-associated small GTPase Rab34. Rab34-positive peri-nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34-induced peri-nuclear lysosome clustering. FLCN interacts directly via its C-terminal DENN domain with the Rab34 effector RILP Using purified recombinant proteins, we show that the FLCN-DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP We propose a model whereby starvation-induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34-positive peri-nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell.

Keywords: BHD syndrome; RILP; Rab34; folliculin; lysosome.

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Figures

Figure 1
Figure 1. FLCN is required for starvation‐dependent peri‐nuclear clustering of lysosomes

  1. Widefield immunofluorescence images of PFA‐fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1‐positive membranes in the peri‐nuclear region. White dotted line shows cell periphery in starved condition. Scale bar, 10 μm.

  2. Quantification of FLCN/LAMP1 association (left) in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100× magnification using a widefield microscope. Data represent 60 cells from 3 independent experiments, error bars show SEM, ***P < 0.001 (two‐tailed t‐test). Western blot (right) shows levels of phosphorylated of S6K and 4EBP in whole‐cell extracts under the same conditions.

  3. Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool. Graph shows relative FLCN expression (n = 3, error bars show ± SEM).

  4. Representative confocal fluorescence images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non‐targeting siRNA. The location of the Golgi (green) is highlighted by giantin staining. Scale bar, 10 μm.

  5. Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution.

  6. Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Error bars show ± SEM from 30 cells in 3 replicates.

  7. Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non‐targeting siRNA or cells depleted of FLCN. Error bars show ± SEM from 30 cells in 3 replicates. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting.

Figure EV1
Figure EV1. A cumulative intensity method for quantification of lysosome distribution

  1. Graph showing cumulative distribution of LAMP1 intensity (left) in GFP, GFP‐RILP or myc‐SKIP transfected HeLa cells. Error bars show ± SEM from 20 cells. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Representative images of GFP‐RILP or myc‐SKIP transfected HeLa cells stained with LAMP1 (right) illustrating application of cumulative intensity distribution method. Scale bar, 10 μm.

  2. Immunofluorescence images and graph showing distribution of LAMP1 in HeLa cells treated for 30 min with Ringer's pH 7.4 or Acetate Ringer's pH 6.5. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Error bars show ± SEM from 3 cells in 3 replicates.

Figure EV2
Figure EV2. Role of the FNIP proteins in control of lysosome distribution

  1. Graphs showing cumulative LAMP1 distribution (top) in cells transfected with non‐targeting siRNA or siRNA against FNIP1/2, in normal growth or starvation conditions. Error bars show ± SEM from 30 cells. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Image of ethidium bromide stained agarose gel (bottom) showing results of RT–PCR experiment with oligos designed to detect FNIP1 and FNIP2 from total RNA preparations using 50 ng of total RNA from cells transfected with siRNA against both proteins.

  2. Confocal immunofluorescence images showing lysosome distribution and localisation of endogenous FLCN in control cells or cells transfected with siRNA against FNIP1/2.

  3. Widefield immunofluorescence image of HA‐FNIP2 transfected HeLa cell showing co‐localisation of endogenous FLCN and HA‐FNIP2.

  4. Western blot of whole‐cell lysates showing relative expression of FLCN‐HA, HA‐FNIP1 and HA‐FNIP2 following transfection in HeLa cells. Blot is over‐exposed for FLCN‐HA to visualise HA‐FNIP1/2.

Figure 2
Figure 2. Distribution and dynamics of FLCN‐GFP in HeLa cells

  1. Representative spinning disc confocal image showing FLCN‐GFP and lysosomes (Lysotracker‐Red) in a live FLCN‐GFP/HA‐FNIP2 transfected HeLa cell (from Movie EV1). Top panels show the first frame of a 180‐frame (1fps) image series. Bottom panels show a maximum intensity projection of the image series to highlight mobile and immobile components. Boxed and magnified regions show typical peri‐nuclear (i) and peripheral (ii) regions in the cell as well as a FLCN‐GFP tubule (iii). Scale bar, 5 μm.

  2. Spinning disc confocal image series of a region live FLCN‐GFP/HA‐FNIP2 transfected HeLa cell highlighting dynamic FLCN‐GFP tubules (Movie EV2). First merge panel shows FLCN‐GFP and Lysotracker‐Red at T = 0. Subsequent panels show only FLCN‐GFP at 30‐s intervals.

  3. Confocal image of a region of a methanol‐fixed FLCN‐GFP/HA‐FNIP2 transfected HeLa cell showing Rab7 (red) and LAMP1 (blue) association with FLCN‐GFP tubules. Scale bar, 1 μm.

Figure 3
Figure 3. FLCN supports Rab34‐dependent peri‐nuclear lysosome clustering

  1. Graphs showing cumulative distribution of LAMP1 intensity in control GFP (black), GFP‐Rab34 (green) or GFP‐Rab7 (blue) transfected cells. Note black line and symbols partially obscure blue. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Error bars show ± SEM from 30 cells in 3 replicates.

  2. GST‐RILP pull‐down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions. Quantification is mean of 3 independent experiments. Error is SEM (two‐tailed t‐test).

  3. Representative confocal fluorescence images of FLCN depleted or control cells, transfected with GFP‐Rab34. Scale bar, 10 μm.

  4. Graphs showing mean GFP‐Rab34 expression (left) and cumulative distribution of LAMP1 intensity (right) in FLCN depleted or control siRNA transfected HeLa cells expressing GFP‐Rab34. Error bars show ± SEM from 30 cells in 3 replicates. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting.

  5. Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 h in HeLa cells. Error bars show SEM from 3 experiments, ***P < 0.001 (two‐tailed t‐test).

  6. Representative confocal fluorescence images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of Rab34 or control cells transfected with a non‐targeting siRNA. Giantin is shown in green. Scale bar, 10 μm.

  7. Graphs showing cumulative distribution of LAMP1 intensity in normal growth or starvation conditions for control cells or cells depleted of Rab34 using siRNA. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting. Error bars show ± SEM from 30 cells in 3 replicates.

  8. Graph showing percentage of LAMP1 in cis‐medial Golgi region (giantin overlap) in the indicated conditions. Error bars show SEM from 30 cells in 3 replicates, ***P < 0.001 (two‐tailed t‐test).

Figure EV3
Figure EV3. GFP‐Rab34 associates with the Golgi
Confocal maximum intensity projection immunofluorescence images of Golgi regions of HeLa cells transfected with GFP‐Rab34 and co‐stained for LAMP1 and GM130 (top), giantin (middle) or golgin‐97 (bottom).
Figure 4
Figure 4. Peri‐nuclear Rab34‐positive membranes contact lysosomes

  1. 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.

  2. 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.

  3. Single confocal sections from the Z‐stack in (B). Orange arrows show close LAMP1/Rab34 association.

  4. 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.

  5. 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.

  6. 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.

  7. Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells. Scale bar, 10 μm.

Figure EV4
Figure EV4. Localisation of endogenous Rab34

  1. Maximum intensity projection images of confocal Z‐stacks showing LAMP1 and endogenous Rab34 localisation in HeLa cells under growth and starvation conditions. Boxes highlight regions in zoom panels in Fig 4B. Scale bar, 10 μm.

  2. Maximum intensity projection images of confocal Z‐stacks under starvation conditions showing LAMP1 and endogenous Rab34 localisation in cells transfected with a non‐targeting siRNA or when FLCN is depleted. Scale bar, 10 μm.

Figure 5
Figure 5. High‐resolution image analysis of Rab34 lysosome contacts
Images from fixed GFP‐Rab34 transfected cells showing contacts between GFP‐Rab34‐positive peri‐nuclear membranes and lysosomes.

  1. Spinning disc confocal microscopy images acquired at 160× magnification. Left panels show a maximum intensity projection imaged of a Z‐stack, sampled according to the Nyquist criterion and deconvolved. Right panels show a 3D volume rendering of the same data. Scale bar, 2 μm.

  2. A projection of 4 planes from a structured illumination microscopy super‐resolution Z‐stack of the same cells. Scale bar, 2 μm.

  3. A thin‐section electron microscopy image of the peri‐nuclear region of a GFP‐Rab34 transfected HeLa cell. Asterisks (*) indicate lysosomes.

  4. A cryo‐immuno‐EM image of a GFP‐Rab34 transfected HeLa cell. GFP‐Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34‐positive membranes. Sites of contact are highlighted by arrows.

Figure EV5
Figure EV5. Targeting of Rab34 and folliculin to mitochondria

  1. Confocal immunofluorescence images of Rab34/Rab35‐dsRED‐Mito transfected HeLa cells showing expected targeting of Rab34‐dsRED‐Mito and Rab35‐dsRED‐Mito to mitochondria (labelled with anti‐mitochondria antibody). Scale bar, 10 μm.

  2. Confocal Immunofluorescence images of HeLa cells transfected with Rab35‐dsRED‐Mito FLCN‐GFP and HA‐FNIP2. White arrows highlight FLCN‐GFP/HA‐FNIP2 co‐localisation.

  3. Confocal immunofluorescence images of Rab34‐dsRED‐Mito (WT or Q111L) and FLCN‐GFP (without HA‐FNIP2) transfected HeLa cells showing recruitment to mitochondria. Scale bar, 10 μm.

  4. SIM super‐resolution image showing a single plane of a region of a Rab34 (Q111L)‐dsRED‐Mito/FLCN‐GFP/HA‐FNIP‐2 transfected cell stained with LAMP1 antibodies. Scale bar, 2 μm.

Figure 6
Figure 6. FLCN directly promotes the association of Rab34 and RILP

  1. Confocal immunofluorescence images of HeLa cells transfected with Rab34‐dsRED‐Mito, FLCN‐GFP and HA‐FNIP2. White arrows highlight FLCN‐GFP/HA‐FNIP2 co‐localisation distinct from Rab34‐mitochondria/FLCN‐GFP localisation. Scale bar, 10 μm.

  2. Confocal immunofluorescence images of Rab34‐dsRED‐Mito (WT) and FLCN‐GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain‐dependent recruitment to mitochondria. Scale bar, 10 μm.

  3. Western blot analysis of GST‐RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.

  4. Western blot analysis of GST‐RILP pull down from UOK257 cells showing DENN domain‐dependent association of FLCN with RILP N‐terminal, Rab binding domain (RBD) and C‐terminal fragments.

  5. Western blot showing binding of His‐tagged FLCN‐DENN domain in E. coli extract to GST‐RILP.

  6. Western blot analysis of GST‐RILP pull down showing direct interaction between RILP and GTP hydrolysis‐deficient (Q111L) Rab34 (at 200 nM, in the presence of 1 mM GTPγS) and FLCN‐DENN domain (1 μM), but not low nucleotide affinity (T66N) Rab34. FLCN‐DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP/Rab34 Q111L binding. Error bars show ± SEM. **P < 0.01 (two‐tailed t‐test).

  7. Western blot analysis of GST‐RILP/Rab34 Q111L pull down (Rab34 at 200 nM) showing that enhanced association of Rab34 with RILP is achieved with low concentrations of FLCN.

  8. Western blot analysis of GST‐RILP pull‐down experiments with GTPγS loaded (in the presence of 1 mM GTPγS) and GDP loaded (in the presence of 1 mM GDP) wild‐type Rab34, showing FLCN‐DENN (500 nM)‐induced interaction with RILP.

  9. Western blot analysis of a GFP‐Trap immunoprecipitation experiment from HeLa cells showing that expression of HA‐RILP promotes the association of GFP‐Rab34 and HA‐Rab7.

Figure 7
Figure 7. Folliculin controls lysosome distribution and dynamics in BHD kidney cancer cells

  1. Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257‐2 cell extracts and relative levels of the active forms as measured by GST‐RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments, error bars show SEM, *P < 0.05 (two‐tailed t‐test).

  2. Representative confocal immunofluorescence images (top) showing intracellular distribution of LAMP1 in UOK257 and UOK257‐2 cells. Dashed line highlights cell periphery in UOK257‐2 cells. Scale bar, 10 μm. Graphs showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257‐2 cells (bottom left) and Lysotracker‐Red PCC decay with time from confocal movies of UOK257 and UOK257‐2 cells (bottom right). Error bars show ± SEM from 30 cells in 3 replicates (left) or from 5 cells (right). P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting.

  3. Widefield immunofluorescence images showing early endosomes (EEA1) and Golgi (golgin‐97) in UOK257 and UOK257‐2 cells. Scale bar, 10 μm.

  4. Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines.

  5. Representative widefield fluorescence images showing intracellular distribution of LAMP1 in UOK257, UOK257‐FLCN and UOK257‐FLCNΔDENN cells. Scale bar, 10 μm. Graph showing cumulative distribution of LAMP1 intensity in UOK257, UOK257‐FLCN and UOK257‐FLCNΔDENN cells. Error bars show ± SEM from 30 cells in 3 replicates. P‐value is determined by the extra sum of F‐squares test following nonlinear regression and curve fitting.

  6. Western blot (left) showing endogenous Rab7 and Rab34 expression in UOK257, UOK257‐FLCN and UOK257‐FLCNΔDENN cells and relative levels of activity as measured by GST‐RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 (right) from 3 independent experiments (*P < 0.05, ***P < 0.001, two‐tailed t‐test).

  7. Western blot showing endogenous RILP expression in the indicated cell lines. Graph shows quantification of RILP expression from 3 independent experiments (*P < 0.05, **P < 0.01, two‐tailed t‐test).

Figure EV6
Figure EV6. In vitro, the FLCN DENN domain does not possess Rab34 GEF activity

  1. Graphs showing results of Rab34 GEF assays; 300 nM Rab34 loaded with mant‐GDP and incubated with various combinations and concentrations of FLCN‐DENN and RILP. GTP was added at a concentration of 0.3 mM or EDTA at a concentration of 10 mM at the 2 min time point. Data were acquired at 10‐s intervals for 25 min. Curves are mean of duplicate samples and are smoothened using a 6‐point rolling average to reduce instrument noise. The same EDTA curve is reproduced across all three graphs for comparison.

  2. Coomassie stained SDS–PAGE gel showing samples of His‐tagged Rab34 WT, Q111L, T66N and FLCN‐DENN domain proteins used in this study.

Figure EV7
Figure EV7. Lysosome dynamics in UOK257 and UOK257‐2 cells
Maximum intensity projection images from 120 frames of Movies EV5 and EV6 highlighting GFP‐Rab7 (green) and Lysotracker‐Red (magenta) dynamics in UOK257 and UOK257‐2 cells during the course of the movie.
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