A deep learning-based tool for the automated detection and analysis of caveolae in transmission electron microscopy images.

Caveolae are nanoscopic and mechanosensitive invaginations of the plasma membrane, essential for adipocyte biology. Transmission electron microscopy (TEM) offers the highest resolution for caveolae visualization, but provides complicated images that are difficult to classify or segment using traditional automated algorithms such as threshold-based methods. As a result, the time-consuming tasks of localization and quantification of caveolae are currently performed manually. We used the Keras library in R to train a convolutional neural network with a total of 36,000 TEM image crops obtained from adipocytes previously annotated manually by an expert. The resulting model can differentiate caveolae from non-caveolae regions with a 97.44% accuracy. The predictions of this model are further processed to obtain caveolae central coordinate detection and cytoplasm boundary delimitation. The model correctly finds negligible caveolae predictions in images from caveolae depleted Cav1-/- adipocytes. In large reconstructions of adipocyte sections, model and human performances are comparable. We thus provide a new tool for accurate caveolae automated analysis that could speed up and assist in the characterization of the cellular mechanical response.

An Abl-FBP17 mechanosensing system couples local plasma membrane curvature and stress fiber remodeling during mechanoadaptation.

Cells remodel their structure in response to mechanical strain. However, how mechanical forces are translated into biochemical signals that coordinate the structural changes observed at the plasma membrane (PM) and the underlying cytoskeleton during mechanoadaptation is unclear. Here, we show that PM mechanoadaptation is controlled by a tension-sensing pathway composed of c-Abl tyrosine kinase and membrane curvature regulator FBP17. FBP17 is recruited to caveolae to induce the formation of caveolar rosettes. FBP17 deficient cells have reduced rosette density, lack PM tension buffering capacity under osmotic shock, and cannot adapt to mechanical strain. Mechanistically, tension is transduced to the FBP17 F-BAR domain by direct phosphorylation mediated by c-Abl, a mechanosensitive molecule. This modification inhibits FBP17 membrane bending activity and releases FBP17-controlled inhibition of mDia1-dependent stress fibers, favoring membrane adaptation to increased tension. This mechanoprotective mechanism adapts the cell to changes in mechanical tension by coupling PM and actin cytoskeleton remodeling.

Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1.

The biology of caveolin-1 (Cav1)/caveolae is intimately linked to actin dynamics and adhesion receptors. Caveolar domains are organized in hierarchical levels of complexity from curved or flattened caveolae to large, higher-order caveolar rosettes. We report that stress fibers controlled by Abl kinases and mDia1 determine the level of caveolar domain organization, which conditions the subsequent inward trafficking of caveolar domains induced upon loss of cell adhesion from the extracellular matrix. Abl-deficient cells have fewer stress fibers, a smaller pool of stress-fiber co-aligned Cav1 and increased clustering of Cav1/caveolae at the cell surface. Defective caveolar linkage to stress fibers prevents the formation of big caveolar rosettes upon loss of cell adhesion, correlating with a lack of inward trafficking. Live imaging of stress fibers and Cav1 showed that the actin-linked Cav1 pool loses its spatial organization in the absence of actin polymerization and is dragged and clustered by depolymerizing filaments. We identified mDia1 as the actin polymerization regulator downstream of Abl kinases that controls the stress-fiber-linked Cav1 pool. mDia1 knockdown results in Cav1/caveolae clustering and defective inward trafficking upon loss of cell adhesion. By contrast, cell elongation imposed by the excess of stress fibers induced by active mDia1 flattens caveolae. Furthermore, active mDia1 rescues the actin co-aligned Cav1 pool and Cav1 inward trafficking upon loss of adhesion in Abl-deficient cells. Thus, caveolar domain organization and trafficking are tightly coupled to adhesive and stress fiber regulatory pathways.

Phosphorylated filamin A regulates actin-linked caveolae dynamics.

Caveolae are relatively stable membrane invaginations that compartmentalize signaling, regulate lipid metabolism and mediate viral entry. Caveolae are closely associated with actin fibers and internalize in response to diverse stimuli. Loss of cell adhesion is known to induce rapid and robust caveolae internalization and trafficking toward a Rab11-positive recycling endosome; however, pathways governing this process are poorly understood. Here, we report that filamin A is required to maintain the F-actin-dependent linear distribution of caveolin-1. High spatiotemporal resolution particle tracking of caveolin-1-GFP vesicles by total internal reflection fluorescence (TIRF) microscopy revealed that FLNa is required for the F-actin-dependent arrest of caveolin-1 vesicles in a confined area and their stable anchorage to the plasma membrane. The linear distribution and anchorage of caveolin-1 vesicles are both required for proper caveolin-1 inwards trafficking. De-adhesion-triggered caveolae inward trafficking towards a recycling endosome is impaired in FLNa-depleted HeLa and FLNa-deficient M2-melanoma cells. Inwards trafficking of caveolin-1 requires both the ability of FLNa to bind actin and cycling PKCα-dependent phosphorylation of FLNa on Ser2152 after cell detachment.