The mechano-sensitive response of β1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein

. Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of a SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, β1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling, and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity. against p18/LAMTOR1 (sh-p18) grown on glass coverslips overnight. YAP subcellular localization was analyzed by confocal microscopy and quantified with the Fiji software. Data are the mean ± SD of 2 independent experiments with n >30 (two - tailed unpaired Student's t -test). resc cells (sh-p18 cells that express p18-GFP) that express or not the active form of SRC (SRC YF ) were seeded overnight on glass coverslips. YAP subcellular localization was quantified from confocal images using the Fiji software. Data are the mean ±SD of 2 independent experiments with >30 cells/condition (two - tailed unpaired Student's t -test).


Abstract.
Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the The nuclear shuttling of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) is directly controlled through ECM compliance and composition and also through cell shape and confluence (8). In the nucleus, YAP and TAZ interact with TEAD family members to drive or modulate gene expression (6). In turn, the expression of specific genes affects cell behavior, such as proliferation/differentiation and migration, thus integrating external cues for cells to adapt to their extracellular environment (9).
Over the last decade, the core signaling pathway leading to YAP and TAZ nuclear translocation has been extensively studied, giving important insights into the cell physiology (9)(10). Integrin-dependent cell adhesion and particularly β1 integrins are crucial for YAP nuclear translocation (11)(12). Indeed, β1 integrin-dependent cell  5G) indicating that the effect of p18 directed sh RNAs was not due to an off target silencing.
In agreement with its reduced nuclear localization, YAP was reduced but hyperphosphorylated in sh-p18 cells compared with sh-ctl and rescued cells (resc) ( Figure   2C). The reduced level of YAP is likely reflecting its phosphorylation dependent degradation as previously reported (27).
As mentioned above the LAMTOR complex docks at LE surfaces using p18/LAMTOR1 and in particular its N-terminal moiety.
Therefore, the addition of a tag to p18/LAMTOR1 N-terminus should interfere with its recruitment to the LE surface and this fusion protein should act as a dominant negative form. Therefore, to complement the silencing strategy, we generated a p18/LAMTOR1 chimeric protein in which the GFP protein was fused to its N terminus (GFP-p18). As expected for a dominant negative construction, GFP-p18 displayed a diffuse staining within the cells and the LAMTOR complex was no longer detected at the LE surface ( Figure S1A and S1B).   Figure 3A).  Figure   3D-E). We also observed that LE vesicles (green in Figure 3E) were in close contact with FAs (red in Figure 3E) in control cells.   In addition to β1 integrins, ILK also has been implicated in YAP signaling (12,33).
One of ILK functions is to scaffold proteins to allow microtubule anchoring to FAs To more directly assess whether the microtubule targeting defect at FAs affected YAP nuclear translocation, we expressed a β3 integrin-ILK-GFP chimeric protein in β1 -/cells to force ILK localization to FAs. The chimeric protein was expressed at similar level than 3-GFP used as control ( Figure   S2E) and correctly incorporated in FAs, as previously reported (35). Microtubule targeting to FAs was restored in β3 integrin-ILK-GFP-, but not in β3 integrin-GFPexpressing cells ( Figure 4F, upper panels).
Immunostaining and quantification of the cytoplasmic/nuclear YAP ratio revealed a significant increase in YAP nuclear translocation in β3 integrin-ILK-GFPexpressing β1 -/cells when compared to β3-GFP-expressing β1 -/or β1-/cells ( Figure   4F, lower panels, and Figure 4G). These data show that microtubule targeting to FAs is crucial for YAP nuclear translocation to control LAMTOR-positive LE targeting to FAs.

p18/LAMTOR1 regulates SRC signaling.
We next hypothesized that these LE vesicles   Figure 5F, figure S3A). In sh-p18 cells, total SRC and pSRC Y416 signal in FAs was strongly increased upon expression of exogenous p18 (resc cells). Incubation with the mTORC1 inhibitor everolimus, which did not have any effect on LE distribution ( Figure 3G), did not modify total SRC and pSRC Y416 signal at FAs ( Figure 5F).
Next, we monitored the phosphorylation and localization of two well established SRC downstream target: p130 CAS and paxillin. In agreement with the reduced level of activated SRC in FAs of sh-p18 cells, p130 CAS phosphorylation also was reduced upon p18/LAMTOR1 silencing ( Figure 5E).
In sh-ctl cells, we observed p130 CAS phosphorylation mainly at FA sites, as expected, whereas it was markedly reduced in sh-p18 ( Figure S3B). As previous studies showed that p130 CAS is phosphorylated at FAs upon stretching these data strongly suggest that p18/LAMTOR1 controls SRC delivery and activity at these sites (38).

CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest with the contents of this article. different only between the 20 kPa and 2 kPa conditions for 0-1 (***) and 1-2 (**) µm (two-tailed unpaired Student's t-test).

G.
Comparison of p18/LAMTOR1-GFP subcellular distribution in control (β1 f/f , green) and in β1-deficient osteoblasts (β1 -/-, blue) cells quantified using the Icy software. Histograms represent the localization from the cell edges to the cell nucleus and is expressed as percentage of all vesicles. The localization of p18/LAMTOR1-positive vesicles was significantly different only for 0-1 (***) and 1-2 (**) µm (two-tailed unpaired Student's t-test).

I.
Comparison of the subcellular distribution of EEA1-positive vesicles in control (β1 f/f , green) and in β1-deficient osteoblasts (β1 -/-, blue) cells quantified using the Icy software.
Histograms represent the localization from the cell edges to the cell nucleus and is expressed as percentage of all vesicles.

A.
Comparison of the YAP cytoplasmic to nuclear ratios (logarithmic scale) in pre-osteoblast cells that stably express mCherry-YAP wt and scramble (sh-ctl) or shRNAs against

C.
Western blot analysis of YAP, YAP pS127 and p18/LAMTOR1 expression in t h e i n d i c a t e d p r e -osteoblast cell lines. Bands intensities were quantified with a Chemidoc CCD camera (Biorad) and ratios were calculated with the Image Lab software (Biorad). Actin was used as internal loading control. Representative of 3 independent experiments. Resc, sh-p18 cells that express exogenous p18/LAMTOR1-GFP.

D.
Comparison of YAP cytoplasmic to nuclear ratios (logarithmic scale) in pre-osteoblast cells (β1 f/f ) that express p18-GFP or GFP-p18 after overnight growth on glass coverslips. YAP subcellular localization was analyzed by indirect immunofluorescence and signal intensity was quantified from confocal images with the Fiji software. Data (mean ± SD) are representative of 2 independent experiments with n >30 cells analyzed (two-tailed unpaired Student's t-test).

A.
Western blot analysis of the ribosomal protein S6 (S6) and its phosphorylated form (pS6).
Bands intensity was quantified with a Chemidoc CCD camera (Biorad) and the Image Lab software (Biorad). Actin was used as internal loading control. Representative of 3 independent experiments.

B.
Comparison of YAP cytoplasmic to nuclear ratio (logarithmic scale) in pre-osteoblasts (β1 f/f ) grown overnight and then incubated (evero.) or not (ctl) with everolimus (10 nM, 3 hours) and stained for YAP. YAP subcellular localization was analyzed by confocal microscopy and intensity values obtained using the Fiji software. Data are the mean ± SD of 2 independent experiments with n >30 cells analyzed (two-tailed unpaired Student's t-test).

D.
Comparison of Rab7-GFP subcellular distribution in sh-ctl (green histograms) and sh-p18 Due to the lack of appropriate tagged p18/LAMTOR1 construct, rescued cells were not investigated in this experiment.

E.
Sh-ctl and sh-p18 cells that stably express mRFP-paxillin (red) were transiently transfected with GFP-Rab-7 (green). 24h post-transfection cells were seeded on glass coverslips and fixed overnight. Due to the lack of appropriate tagged p18/LAMTOR1 construct, rescued cells were not investigated. Scale bar: 10µm.

F.
Quantification of GFP-Rab-7 targeting to focal adhesions in control (sh-ctl, red) and sh-

G.
Comparison of YAP cytoplasmic to nuclear ratio (logarithmic scale) in β1 f/f and β1 -/preosteoblast that stably express or not the β3-GFP or β3-ILK-GFP fusion protein after overnight growth on glass coverslips. YAP subcellular localization was quantified from confocal microscopy images with the Fiji software; data (mean ± SD) from 2 independent experiments (n >30 cells/condition) (two-tailed unpaired Student's t-test).

A.
Control pre-osteoblast cells were transiently transfected with p18-GFP and SRC-mCherry. 24h post-transfection, cells were seeded on glass coverslips and GFP and mCherry signals acquired by confocal microscopy. Scale bar: 10µm.

B.
Histogram representing the Pearson coefficient and thresholded Manders (tM1, tM2) values obtained from confocal images of p18-GFP and SRC-mCherry co-transfected pre-Subcellular localization of YAP in sh-p18 pre-osteoblast cells and in sh-p18 cells that express constitutively active SRC YF (sh-p18+SRC YF ). Scale bar: 10µm.