Spoiled for Choice: Diverse Endocytic Pathways Function at the Cell Surface.

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.

Post-injury hydraulic fracturing drives fissure formation in the zebrafish basal epidermal cell layer.

The skin epithelium acts as the barrier between an organism's internal and external environments. In zebrafish and other freshwater organisms, this barrier function requires withstanding a large osmotic gradient across the epidermis. Wounds breach this epithelium, causing a large disruption to the tissue microenvironment due to the mixing of isotonic interstitial fluid with the external hypotonic fresh water. Here, we show that, following acute injury, the larval zebrafish epidermis undergoes a dramatic fissuring process that resembles hydraulic fracturing, driven by the influx of external fluid. After the wound has sealed-preventing efflux of this external fluid-fissuring starts in the basal epidermal layer at the location nearest to the wound and then propagates at a constant rate through the tissue, spanning over 100 µm. During this process, the outermost superficial epidermal layer remains intact. Fissuring is completely inhibited when larvae are wounded in isotonic external media, suggesting that osmotic gradients are required for fissure formation. Additionally, fissuring partially depends on myosin II activity, as myosin II inhibition reduces the distance of fissure propagation away from the wound. During and after fissuring, the basal layer forms large macropinosomes (with cross-sectional areas ranging from 1 to 10 µm2). We conclude that excess external fluid entry through the wound and subsequent closure of the wound through actomyosin purse-string contraction in the superficial cell layer causes fluid pressure buildup in the extracellular space of the zebrafish epidermis. This excess fluid pressure causes tissue to fissure, and eventually the fluid is cleared through macropinocytosis.

Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis.

Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission.

Exploiting cell-to-cell variability to detect cellular perturbations.

Any single-cell-resolved measurement generates a population distribution of phenotypes, characterized by a mean, a variance, and a shape. Here we show that changes in the shape of a phenotypic distribution can signal perturbations to cellular processes, providing a way to screen for underlying molecular machinery. We analyzed images of a Drosophila S2R+ cell line perturbed by RNA interference, and tracked 27 single-cell features which report on endocytic activity, and cell and nuclear morphology. In replicate measurements feature distributions had erratic means and variances, but reproducible shapes; RNAi down-regulation reliably induced shape deviations in at least one feature for 1072 out of 7131 genes surveyed, as revealed by a Kolmogorov-Smirnov-like statistic. We were able to use these shape deviations to identify a spectrum of genes that influenced cell morphology, nuclear morphology, and multiple pathways of endocytosis. By preserving single-cell data, our method was even able to detect effects invisible to a population-averaged analysis. These results demonstrate that cell-to-cell variability contains accessible and useful biological information, which can be exploited in existing cell-based assays.