Getting cells into shape by calcium-dependent actin cross-linking proteins.

The actin cytoskeleton represents a highly dynamic filament system providing cell structure and mechanical forces to drive a variety of cellular processes. The dynamics of the actin cytoskeleton are controlled by a number of conserved proteins that maintain the pool of actin monomers, promote actin nucleation, restrict the length of actin filaments and cross-link filaments into networks or bundles. Previous work has been established that cytoplasmic calcium is an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we summarize new recent perspectives on how calcium fluxes are transduced to the actin cytoskeleton in a physiological context. In this mini-review we will focus on three calcium-binding EF-hand-containing actin cross-linking proteins, α-actinin, plastin and EFHD2/Swiprosin-1, and how these conserved proteins affect the cell's actin reorganization in the context of cell migration and wound closure in response to calcium.

Swip-1 promotes exocytosis of glue granules in the exocrine Drosophila salivary gland.

Exocytosis is a fundamental cellular process by which cells secrete cargos from their apical membrane into the extracellular lumen. Cargo release proceeds in sequential steps that depend on coordinated assembly and organization of an actin cytoskeletal network. Here, we identified the conserved actin-crosslinking protein Swip-1 as a novel regulator controlling exocytosis of glue granules in the Drosophila salivary gland. Real-time imaging revealed that Swip-1 is simultaneously recruited with F-actin onto secreting granules in proximity to the apical membrane. We observed that Swip-1 is rapidly cleared at the point of secretory vesicle fusion and colocalizes with actomyosin network around the fused vesicles. Loss of Swip-1 function impairs secretory cargo expulsion, resulting in strongly delayed secretion. Thus, our results uncover a novel role of Swip-1 in secretory vesicle compression and expulsion of cargo during regulated exocytosis. Remarkably, this function neither requires Ca2+ binding nor dimerization of Swip-1. Our data rather suggest that Swip-1 regulates actomyosin activity upstream of Rho-GTPase signaling to drive proper vesicle membrane crumpling and expulsion of cargo.

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.

Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.

S-Sulfocysteine Induces Seizure-Like Behaviors in Zebrafish.

Sulfite is a neurotoxin, which is detoxified by the molybdenum cofactor (Moco)-dependent enzyme sulfite oxidase (SOX). In humans, SOX deficiency causes the formation of the glutamate analog S-Sulfocysteine (SSC) resulting in a constant overstimulation of ionotropic glutamatergic receptors. Overstimulation leads to seizures, severe brain damage, and early childhood death. SOX deficiency may be caused either by a mutated sox gene or by mutations in one of the genes of the multi-step Moco biosynthesis pathway. While patients affected in the first step of Moco biosynthesis can be treated by a substitution therapy, no therapy is available for patients affected either in the second or third step of Moco biosynthesis or with isolated SOX deficiency. In the present study, we used a combination of behavior analysis and vital dye staining to show that SSC induces increased swimming, seizure-like movements, and increased cell death in the central nervous system of zebrafish larvae. Seizure-like movements were fully revertible upon removal of SSC or could be alleviated by a glutamatergic receptor antagonist. We conclude that in zebrafish SSC can chemically induce phenotypic characteristics comparable to the disease condition of human patients lacking SOX activity.

Culture and Transfection of Zebrafish Primary Cells.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue. In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.

Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.