Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (deep picker in context), an open-source deep-learning framework for supervised segmentation and macromolecular complex localization in cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles, and cytosol. By comparing DeePiCt to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, applying pre-trained networks to a HeLa cell tomogram demonstrates that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate use by the community.

Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction-force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. Our experimental results and modeling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignement and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.

BackgroundThe navigation of magnetotactic bacteria relies on specific intracellular organelles, the magnetosomes, which are membrane-enclosed crystals of magnetite aligned into a linear chain. The magnetosome chain acts as a cellular compass, aligning the cells in the geomagnetic field in order to search for suitable environmental conditions in chemically stratified water columns and sediments. During cytokinesis, magnetosome chains have to be properly positioned, cleaved and separated in order to be evenly passed into daughter cells. In Magnetospirillum gryphiswaldense, the assembly of the magnetosome chain is controlled by the actin-like MamK, which polymerizes into cytoskeletal filaments that are connected to magnetosomes through the acidic MamJ protein. MamK filaments were speculated to recruit the magnetosome chain to cellular division sites, thus ensuring equal organelle inheritance. However, the underlying mechanism of magnetic organelle segregation has remained largely unknown.ResultsHere, we performed in vivo time-lapse fluorescence imaging to directly track the intracellular movement and dynamics of magnetosome chains as well as photokinetic and ultrastructural analyses of the actin-like cytoskeletal MamK filament. We show that magnetosome chains undergo rapid intracellular repositioning from the new poles towards midcell into the newborn daughter cells, and the driving force for magnetosomes movement is likely provided by the pole-to-midcell treadmilling growth of MamK filaments. We further discovered that splitting and equipartitioning of magnetosome chains occurs with unexpectedly high accuracy, which depends directly on the dynamics of MamK filaments.ConclusionWe propose a novel mechanism for prokaryotic organelle segregation that, similar to the type-II bacterial partitioning system of plasmids, relies on the action of cytomotive actin-like filaments together with specific connectors, which transport the magnetosome cargo in a fashion reminiscent of eukaryotic actin-organelle transport and segregation mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0290-1) contains supplementary material, which is available to authorized users.