Lysosomes are highly dynamic organelles that regulate metabolic signaling pathways by recruiting cytosolic proteins to platforms on their limiting membrane. To identify new proteins recruited to these platforms, we performed a proximity-dependent labelling screen using as bait a component of the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR) complex, which regulates lysosome positioning and mTORC1, AMPK and MEK/ERK signaling. We identified a network of proteins involved in actin remodelling, the most prominent of which was Pleckstrin homology domain-containing family G member 3 (PLEKHG3), a Rho guanine nucleotide exchange factors that binds to actin filaments and is enriched in protrusions. We show that GFP-PLEKHG3 accumulates in focal adhesion sites, where it colocalizes with peripheral lysosomes. Peripheral accumulation of lysosomes concentrates PLEKHG3 below the plasma membrane, inhibits protrusion formation and limits cell motility. This study reveals that the subcellular positioning of lysosomes plays a role in the intracellular distribution of PLEKHG3 and in the cell’s protrusion activity, shape, and motility.

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.
© 2021 Rabanal-Ruiz et al.

Migration-inducing gene 7 (MIG7) is highly expressed and is implicated in multiple malignant tumors with vasculogenic mimicry (VM) which renders possible routes without the endothelium for invasion and metastasis. However, there are few reports in the literature describing the relationship between MIG7 expression and VM formation in hepatocellular carcinoma (HCC). In the present study, we found a significantly positive correlation between MIG7 expression and VM in 40 HCC specimens. Three-dimensional (3D) culture showed that VM formation in the HCC cell line MHCC-97H with high metastatic potential was enhanced to a greater extent than that of MHCC-97L and Huh-7 with low and non-metastatic potential. There was no VM formation in human normal hepatocyte line L-02. Moreover, MIG7 expression was higher in MHCC-97H than in MHCC-97L and Huh-7 cells and non-detectable in L-02 cells. MIG7 knockdown in MHCC-97H cells reduced VM formation, and weakened the invasive properties accompanying the enhanced cellular adhesion. Notably, there was no significant effect of endostatin (ES), a broad-spectrum angiogenesis inhibitor applied to clinical treatment, on both MIG7 expression and VM formation. Thus, the present study presents a causal link between MIG7 expression and VM formation in HCC, suggesting a potential treatment target for invasion and metastasis.

Integrin adhesome proteins bind each other in alternative manners, forming within the cell diverse cell-matrix adhesion sites with distinct properties. An intriguing question is how such modular assembly of adhesion sites is achieved correctly solely by self-organization of their components. Here we address this question using high-throughput multiplexed imaging of eight proteins and two phosphorylation sites in a large number of single focal adhesions. We found that during the assembly of focal adhesions the variances of protein densities decrease while the correlations between them increase, suggesting reduction in the noise levels within these structures. These changes correlate independently with the area and internal density of focal adhesions, but not with their age or shape. Artificial neural network analysis indicates that a joint consideration of multiple components improves the predictability of paxillin and zyxin levels in internally dense focal adhesions. This suggests that paxillin and zyxin densities in focal adhesions are fine-tuned by integrating the levels of multiple other components, thus averaging-out stochastic fluctuations. Based on these results we propose that increase in internal protein densities facilitates noise suppression in focal adhesions, while noise suppression enables their stable growth and further density increase-hence forming a feedback loop giving rise to a quality-controlled assembly.

In normal hearts, capillaries are densely distributed throughout the myocardial tissue, and the cross-talk between myocytes and capillary endothelial cells plays a pivotal role in regulating cardiac development, maturation and function. Although previous studies have suggested a role for the endothelium in the organisation of nearby cardiomyocytes, the underlying mechanism has yet to be illustrated.
Using a transwell coculture system, we studied the paracrine effect of endothelial cells on cardiomyocytes and found that the regulation of cardiomyocyte spatial reorganisation and cytoskeletal dynamics by endothelial cells was coupled with β1-integrin induction. To determine the role of β1-integrin in this process, we preincubated myocytes with a β1-integrin function-blocking antibody before coculture. β1-integrin blockage abolished myocyte chemotactic activity and inhibited microtubule extension and stress fibre assembly. We further evaluated the therapeutic potential of combined endothelial cell-cardiac myocyte transplantation against ischemic cardiomyopathy in an acute myocardial infarction (AMI) mouse model. The results showed that myocytes and endothelial cells synergistically promoted ischemic myocardial repair, as evidenced by the robust engraftment and migration of implanted cells within the infarcted area, as well as the stimulation of angiogenesis, the attenuation of scar tissue and the improvement of cardiac function.
Our study demonstrated the necessity of β1-integrin in the interactions between cardiomyocytes and endothelial cells and presented a novel combined transplantation approach that might hold promise for treating ischemic cardiomyopathy.
© 2015 S. Karger AG, Basel.