Rapid Synthesis of Cryo-ET Data for Training Deep Learning Models.

Deep learning excels at cryo-tomographic image restoration and segmentation tasks but is hindered by a lack of training data. Here we introduce cryo-TomoSim (CTS), a MATLAB-based software package that builds coarse-grained models of macromolecular complexes embedded in vitreous ice and then simulates transmitted electron tilt series for tomographic reconstruction. We then demonstrate the effectiveness of these simulated datasets in training different deep learning models for use on real cryotomographic reconstructions.

Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in Cells.

Fluorescence microscopy of cytoskeletal proteins in situ using immunolabeling, fluorescent reagents, or expression of tagged proteins has been a common practice for decades but often with too little regard for what might not be visualized. This is especially true for assembled filamentous actin (F-actin), for which binding of fluorescently labeled phalloidin is taken as the gold standard for its quantification even though it is well known that F-actin saturated with cofilin (cofilactin) binds neither fluorescently labeled phalloidin nor genetically encoded F-actin reporters, such as LifeAct. Here, using expressed fluorescent cofilactin reporters, we show that cofilactin is the major component of some actin-containing structures in both normal and stressed neurons and present various fixation, permeabilization, and cryo-preservation methods for optimizing its observation.

Cofilactin filaments regulate filopodial structure and dynamics in neuronal growth cones.

Cofilin is best known for its ability to sever actin filaments and facilitate cytoskeletal recycling inside of cells, but at higher concentrations in vitro, cofilin stabilizes a more flexible, hyper-twisted state of actin known as "cofilactin". While this filament state is well studied, a structural role for cofilactin in dynamic cellular processes has not been observed. With a combination of cryo-electron tomography and fluorescence imaging in neuronal growth cones, we observe that filopodial actin filaments switch between a fascin-linked and a cofilin-decorated state, and that cofilactin is associated with a variety of dynamic events within filopodia.

Challenges and triumphs in cryo-electron tomography.

Cryo-electron tomography has stepped fully into the spotlight. Enthusiasm is high. Fortunately for us, this is an exciting time to be a cryotomographer, but there is still a way to go before declaring victory.

Circadian clock protein BMAL1 regulates melanogenesis through MITF in melanoma cells.

Solar ultraviolet B radiation (UVB) is one of the leading causes of various skin conditions, including photoaging, sunburn erythema, and melanoma. As a protective response, the skin has inbuilt defense mechanisms, including DNA repair, cell cycle, apoptosis, and melanin synthesis. Though DNA repair, cell cycle, and apoptosis are clock controlled, the circadian mechanisms associated with melanin synthesis are not well understood.

Cryo-Electron Tomography and Automatic Segmentation of Cultured Hippocampal Neurons.

Cryo-electron tomography is fast becoming a preferred method for studying intracellular environments at the molecular scale. Increases in data collection throughput means that large numbers of tomograms can be generated at rates too fast for humans to easily explore quantitatively. Currently, there is a large effort to make data collection and segmentation tools more automated.