The primary cilium protein folliculin is part of the autophagy signaling pathway to regulate epithelial cell size in response to fluid flow

Abstract

Autophagy is a conserved molecular pathway directly involved in the degradation and recycling of intracellular components. Autophagy is associated with a response to stress situations, such as nutrients deficit, chemical toxicity, mechanical stress or microbial host defense. We have recently shown that primary cilium-dependent autophagy is important to control kidney epithelial cell size in response to fluid flow induced shear stress. Here we show that the ciliary protein folliculin (FLCN) actively participates to the signaling cascade leading to the stimulation of fluid flow-dependent autophagy upstream of the cell size regulation in HK2 kidney epithelial cells. The knockdown of FLCN induces a shortening of the primary cilium, inhibits the activation of AMPK and the recruitment of the autophagy protein ATG16L1 at the primary cilium. Altogether, our results suggest that FLCN is essential in the dialog between autophagy and the primary cilium in epithelial cells to integrate shear stress-dependent signaling.

Keywords: autophagy; fluid flow; folliculin; primary cilium; shear stress.

Figure 1. FIGURE 1: Shear stress induces autophagy, cell size decrease and FLCN expression. (A-D)

HK2 cells were subjected to fluid flow from 4 h to 4 days (shear 4 h, 48 h, 96h-4D), or not (static 4 h, 48 h, 96h-4D). (A) After fluid flow treatment or static culture, cells were fixed, labeled with DAPI and immunostained for the autophagosome marker LC3 or stained with phalloidin to reveal F-actin and cell boarders are marked out with white dashes (B) and then analyzed by fluorescence microscopy. (C) Quantification of LC3 puncta (LC3 dots number per 500 μm² area) from experiments shown in (A). (D) Quantification of cell area (mean) from experiments shown in (C). (E-F) HK2 cells were subjected to fluid flow from 4 h to 4 days (shear 4 h, 48 h, 96h-4D), or not (static 4 h, 48 h, 96h-4D). (E) Representative western blot analysis of FLCN, LC3I, LC3II and actin in the indicated conditions. (F) Quantification of Western blot shown in (E). (G) HK2 cells were subjected to 4 days (shear 4D) fluid flow or not (static 4D) and FLCN mRNA expression level was determined and quantified by RT-qPCR. (H) HK2 cells were maintained in normal culture condition (CTRL) or subjected to a 24 h serum starvation (SS 24 h). Levels of the FLCN, LC3I and LC3II were analyzed by western blot. Scale bars in (A) and (C) = 10μm.

Figure 2. FIGURE 2: FLCN is associated with primary cilium and ciliogenesis.

(A) HK2 cells grown for 9 6h in static conditions were fixed with methanol, labeled with DAPI, immunostained for FLCN, ARL13B (to reveal primary cilium) and then analyzed by fluorescence microscopy. (B-E) HK2 cells were transfected either with siRNA targeting FLCN (siFLCN) or control siRNA (siCTRL). 72 h later they were subjected to 4 days fluid flow (shear 4D) or not (static 4D). (B) FLCN downregulation by siRNA efficiency was verified by western blot. (C) Cells were fixed with methanol, labeled with DAPI, immunostained for ARL13B (to reveal primary cilium) and then analyzed by fluorescence microscopy. (D, E) Quantification of ciliated cells number and cilia length from experiments shown in (C). Scale bars in (A) = 10μm and (C) = 5μm.

Figure 3. FIGURE 3: FLCN, but not FNIP1, is required for shear stress-induced autophagy.

(A-D) HK2 cells were transfected either with siRNA targeting FLCN (siFLCN) or control siRNA (siCTRL) 72 h later they were subjected to fluid flow (shear) for the indicated times or not (static). (A) Levels of FLCN, LC3I and LC3II were analyzed by western blot and LC3 II/actin ratio was quantified (B). (C) Cells were fixed, labeled with DAPI, immunostained for LC3 and then analyzed by fluorescence microscopy. (D) LC3 dots were quantified from experiments shown in (C). (E-H) HK2 cells were transfected either with siRNA targeting FNIP1 (siFNIP1) or control siRNA (siCTRL). 72 h later they were subjected to fluid flow for 4 days (shear 4D) or not (static 4D). (E) Levels of FNIP1, FLCN, LC3I and LC3II were analyzed by western blot and LC3 II/actin ratio was quantified (F). (G) Cells were fixed, labeled with DAPI, immunostained for LC3 and then analyzed by fluorescence microscopy. (H) LC3 dots were quantified from experiments shown in (G). Scale bars in (C) and (G) = 5μm.

Figure 4. FIGURE 4: FLCN, but not FNIP1, is required for shear stressinduced cell size regulation.

(A-B) HK2 cells were transfected either with siRNA targeting FLCN (siFLCN) or control siRNA (siCTRL) 72 h later they were subjected to fluid flow for 4 days (shear 4D) or not (static 4D). (A) Cells were fixed, labeled with DAPI and phalloidin to reveal F-actin and cell boarder and then analyzed by fluorescence microscopy. (B) Cells areas were quantified from experiments shown in (A). (C-D) HK2 cells were transfected with siRNA targeting FNIP (siF-NIP1) or control siRNA (siCTRL)). The experiments were performed and quantified as in (A-B). Scale bars in (A) and (C) = 10μm.

Figure 5. FIGURE 5: Shear-stress-induced autophagy and cell size regulation are abolished in FLCN-null cells.

UOK 257 FLCN-null cells and UOK 257-2 FLCN restored cells were cultured on microslides and then subjected to fluid flow for 4 days (shear) or not (static 4D). Cells were fixed, labeled with DAPI, immunostained for LC3 (A, C) or labeled with DAPI and immunostained for β-catenin to reveal cell boarder (E, G) and then analyzed by fluorescence microscopy. (B, D) LC3 dots were quantified from experiments shown in (A) (UOK 257cells) and (C) (UOK 257-2 cells). (F, H) cells areas were quantified from experiments shown in (E) (UOK 257cells) and (G) (UOK 257-2 cells). Scale bars in (A, C, E and F) = 10μm.

Figure 6. FIGURE 6: Shear-stress dependent recruitment of ATG16L1 to primary cilium is impaired in FLCN knockdown cells.

HK2 cells were transfected with control siRNA (siCTRL) or with siRNA targeting FLCN (siFLCN). 72 h later, they were subjected to fluid flow for 4 days (shear 4D) or not (static 4D). (A) ATG16L1 and actin levels were analyzed by western blot. (B) Cells prone to fluid flow were fixed with methanol, labeled with DAPI, immunostained for ATG16L1 and ARL13B and then analyzed by fluorescence microscopy. Arrowhead indicates presence of ATG16L1 at the basal body. (C) ATG16L1 positive structures at basal body were quantified from experiments shown in (B). Scale bar = 10μm.

Figure 7. FIGURE 7: FLCN is associated with phospho-AMPK and LKBI mobilization during shear stress.

HK2 cells were transfected with control siRNA (siCTRL) or with siRNA targeting FLCN (siFLCN). 72 h later, they were subjected (shear) or not (static) to fluid flow for the indicated times. (A) Phospho-AMPK (Thr172), total AMPK, FLCN and actin levels were analyzed by western blot and quantified (B) in the indicated conditions (A). (C, D) Cells prone to fluid flow (shear 4D) or not (static 4D) were fixed with methanol, labeled with DAPI, immunostained for ARL13B, phospho-AMPK (C), LKB1 (D), ARL13B, and then analyzed by fluorescence microscopy. Arrowheads indicate presence of phospho-AMPK (P-AMPK) or LKB1 at primary cilium. Scale bars in (C) and (D) = 10μm.