Convolutional networks for supervised mining of molecular patterns within cellular context.

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.

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies.

Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery.

A bacterial cytolinker couples positioning of magnetic organelles to cell shape control.

Magnetotactic bacteria maneuver within the geomagnetic field by means of intracellular magnetic organelles, magnetosomes, which are aligned into a chain and positioned at midcell by a dedicated magnetosome-specific cytoskeleton, the "magnetoskeleton." However, how magnetosome chain organization and resulting magnetotaxis is linked to cell shape has remained elusive. Here, we describe the cytoskeletal determinant CcfM (curvature-inducing coiled-coil filament interacting with the magnetoskeleton), which links the magnetoskeleton to cell morphology regulation in Magnetospirillum gryphiswaldense Membrane-anchored CcfM localizes in a filamentous pattern along regions of inner positive-cell curvature by its coiled-coil motifs, and independent of the magnetoskeleton. CcfM overexpression causes additional circumferential localization patterns, associated with a dramatic increase in cell curvature, and magnetosome chain mislocalization or complete chain disruption. In contrast, deletion of ccfM results in decreased cell curvature, impaired cell division, and predominant formation of shorter, doubled chains of magnetosomes. Pleiotropic effects of CcfM on magnetosome chain organization and cell morphology are supported by the finding that CcfM interacts with the magnetoskeleton-related MamY and the actin-like MamK via distinct motifs, and with the cell shape-related cytoskeleton via MreB. We further demonstrate that CcfM promotes motility and magnetic alignment in structured environments, and thus likely confers a selective advantage in natural habitats of magnetotactic bacteria, such as aquatic sediments. Overall, we unravel the function of a prokaryotic cytoskeletal constituent that is widespread in magnetic and nonmagnetic spirilla-shaped Alphaproteobacteria.

Stress fibres are embedded in a contractile cortical network.

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 modelling 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 alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.

A gradient-forming MipZ protein mediating the control of cell division in the magnetotactic bacterium Magnetospirillum gryphiswaldense.

Cell division needs to be tightly regulated and closely coordinated with other cellular processes to ensure the generation of fully viable offspring. Here, we investigate division site placement by the cell division regulator MipZ in the alphaproteobacterium Magnetospirillum gryphiswaldense, a species that forms linear chains of magnetosomes to navigate within the geomagnetic field. We show that M. gryphiswaldense contains two MipZ homologs, termed MipZ1 and MipZ2. MipZ2 localizes to the division site, but its absence does not cause any obvious phenotype. MipZ1, by contrast, forms a dynamic bipolar gradient, and its deletion or overproduction cause cell filamentation, suggesting an important role in cell division. The monomeric form of MipZ1 interacts with the chromosome partitioning protein ParB, whereas its ATP-dependent dimeric form shows non-specific DNA-binding activity. Notably, both the dimeric and, to a lesser extent, the monomeric form inhibit FtsZ polymerization in vitro. MipZ1 thus represents a canonical gradient-forming MipZ homolog that critically contributes to the spatiotemporal control of FtsZ ring formation. Collectively, our findings add to the view that the regulatory role of MipZ proteins in cell division is conserved among many alphaproteobacteria. However, their number and biochemical properties may have adapted to the specific needs of the host organism.

In Vivo Coating of Bacterial Magnetic Nanoparticles by Magnetosome Expression of Spider Silk-Inspired Peptides.

Magnetosomes are natural magnetic nanoparticles with exceptional properties that are synthesized in magnetotactic bacteria by a highly regulated biomineralization process. Their usability in many applications could be further improved by encapsulation in biocompatible polymers. In this study, we explored the production of spider silk-inspired peptides on magnetosomes of the alphaproteobacterium Magnetospirillum gryphiswaldense. Genetic fusion of different silk sequence-like variants to abundant magnetosome membrane proteins enhanced magnetite biomineralization and caused the formation of a proteinaceous capsule, which increased the colloidal stability of isolated particles. Furthermore, we show that spider silk peptides fused to a magnetosome membrane protein can be used as seeds for silk fibril growth on the magnetosome surface. In summary, we demonstrate that the combination of two different biogenic materials generates a genetically encoded hybrid composite with engineerable new properties and enhanced potential for various applications.

Segregation of prokaryotic magnetosomes organelles is driven by treadmilling of a dynamic actin-like MamK filament.

BACKGROUND: The 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. RESULTS: Here, 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. CONCLUSION: We 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.

In vivo Architecture of the Polar Organizing Protein Z (PopZ) Meshwork in the Alphaproteobacteria Magnetospirillum gryphiswaldense and Caulobacter crescentus.

The polar organizing protein Z (PopZ) forms a polar microdomain that is inaccessible to larger macromolecules such as ribosomes, and selectively sequesters proteins crucial for cell cycle control and polar morphogenesis in various Alphaproteobacteria. However, the in vivo architecture of this microdomain has remained elusive. Here, we analyzed the three-dimensional ultrastructural organization of the PopZ network in Magnetospirillum gryphiswaldense and Caulobacter crescentus by Volta phase plate cryo-electron tomography, which provides high spatial resolution and improved image contrast. Our results suggest that PopZ forms a porous network of disordered short, flexible, and branching filaments.

High-Yield Production, Characterization, and Functionalization of Recombinant Magnetosomes in the Synthetic Bacterium Rhodospirillum rubrum "magneticum".

Recently, the photosynthetic Rhodospirillum rubrum has been endowed with the ability of magnetosome biosynthesis by transfer and expression of biosynthetic gene clusters from the magnetotactic bacterium Magnetospirillum gryphiswaldense. However, the growth conditions for efficient magnetite biomineralization in the synthetic R. rubrum "magneticum", as well as the particles themselves (i.e., structure and composition), have so far not been fully characterized. In this study, different cultivation strategies, particularly the influence of temperature and light intensity, are systematically investigated to achieve optimal magnetosome biosynthesis. Reduced temperatures ≤16 °C and gradual increase in light intensities favor magnetite biomineralization at high rates, suggesting that magnetosome formation might utilize cellular processes, cofactors, and/or pathways that are linked to photosynthetic growth. Magnetosome yields of up to 13.6 mg magnetite per liter cell culture are obtained upon photoheterotrophic large-scale cultivation. Furthermore, it is shown that even more complex, i.e., oligomeric, catalytically active functional moieties like enzyme proteins can be efficiently expressed on the magnetosome surface, thereby enabling the in vivo functionalization by genetic engineering. In summary, it is demonstrated that the synthetic R. rubrum "magneticum" is a suitable host for high-yield magnetosome biosynthesis and the sustainable production of genetically engineered, bioconjugated magnetosomes.

The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy.

Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

The Polar Organizing Protein PopZ Is Fundamental for Proper Cell Division and Segregation of Cellular Content in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria (MTB) are of special scientific interest due to the formation of magnetosomes, intracellular membrane-enveloped magnetite crystals arranged into a linear chain by a dedicated cytoskeleton. Magnetotaxis relies on the formation and proper inheritance of these unique magnetic organelles, both of which need to be coordinated with the segregation of other cellular content such as chromosomes or motility and chemotaxis related structures. Thus, elaborated mechanisms are required in MTB to coordinate and maintain a high level of spatial and temporal subcellular organization during cytokinesis. However, thus far, underlying mechanisms and polarity determinants such as landmark proteins remained obscure in MTB. Here, we analyzed an ortholog of the polar organizing protein Z in the alphaproteobacterium Magnetospirillum gryphiswaldense termed PopZ Mgr We show that deletion of the popZMgr gene causes abnormal cell elongation, minicell formation, DNA missegregation, and impairs motility. Overproduction of PopZ Mgr results in PopZ-rich regions near the poles, which are devoid of larger macromolecules, such as ribosomes, chromosomal DNA, and polyhydroxybutyrate (PHB) granules. Using superresolution microscopy, we show that PopZ Mgr exhibits a bipolar localization pattern throughout the cell cycle, indicating that the definition of new poles in M. gryphiswaldense occurs immediately upon completion of cytokinesis. Moreover, substitution of PopZ orthologs between M. gryphiswaldense and the related alphaproteobacterium Caulobacter crescentus indicated that PopZ localization depends on host-specific cues and that both orthologs have diverged to an extent that allows only partial reciprocal functional complementation. Altogether, our results indicate that in M. gryphiswaldense, PopZ plays a critical role during cell division and segregation of cellular content.IMPORTANCE Magnetotactic bacteria (MTB) share the unique capability of magnetic navigation, one of the most complex behavioral responses found in prokaryotes, by means of magnetosomes, which act as an internal compass. Due to formation of these unique nanoparticles, MTB have emerged as a model to study prokaryotic organelle formation and cytoskeletal organization in conjunction with complex motility systems. Despite the high degree of subcellular organization required in MTB, less is known about cell-cycle-related factors or proteins responsible for spatiotemporal polarity control. Here, we investigate the function of the polar organizer PopZ in the magnetotactic alphaproteobacterium Magnetospirillum gryphiswaldense Although PopZ is widely distributed among the alphaproteobacteria, its function in MTB belonging to this class has remained unexplored. Our results suggest that in M. gryphiswaldense, PopZ has a key role during cell division and subcellular organization. Furthermore, we show that PopZ localization and function differ from other nonmagnetotactic alphaproteobacterial model organisms.

MamY is a membrane-bound protein that aligns magnetosomes and the motility axis of helical magnetotactic bacteria.

To navigate within the geomagnetic field, magnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membrane-enveloped magnetite nanocrystals. In magnetotactic spirilla, magnetosomes become actively organized into chains by the filament-forming actin-like MamK and the adaptor protein MamJ, thereby assembling a magnetic dipole much like a compass needle. However, in Magnetospirillum gryphiswaldense, discontinuous chains are still formed in the absence of MamK. Moreover, these fragmented chains persist in a straight conformation indicating undiscovered structural determinants able to accommodate a bar magnet-like magnetoreceptor in a helical bacterium. Here, we identify MamY, a membrane-bound protein that generates a sophisticated mechanical scaffold for magnetosomes. MamY localizes linearly along the positive inner cell curvature (the geodetic cell axis), probably by self-interaction and curvature sensing. In a mamY deletion mutant, magnetosome chains detach from the geodetic axis and fail to accommodate a straight conformation coinciding with reduced cellular magnetic orientation. Codeletion of mamKY completely abolishes chain formation, whereas on synthetic tethering of magnetosomes to MamY, the chain configuration is regained, emphasizing the structural properties of the protein. Our results suggest MamY is membrane-anchored mechanical scaffold that is essential to align the motility axis of magnetotactic spirilla with their magnetic moment vector and to perfectly reconcile magnetoreception with swimming direction.

Identification and Initial Characterization of Prophages in Vibrio campbellii.

Phages are bacteria targeting viruses and represent the most abundant biological entities on earth. Marine environments are exceptionally rich in bacteriophages, harboring a total of 4x10(30) viruses. Nevertheless, marine phages remain poorly characterized. Here we describe the identification of intact prophage sequences in the genome of the marine γ-proteobacterium Vibrio campbellii ATCC BAA-1116 (formerly known as V. harveyi ATCC BAA-1116), which presumably belong to the family of Myoviridae. One prophage was found on chromosome I and shows significant similarities to the previously identified phage ΦHAP-1. The second prophage region is located on chromosome II and is related to Vibrio phage kappa. Exposure of V. campbellii to mitomycin C induced the lytic cycle of two morphologically distinct phages and, as expected, extracellular DNA from induced cultures was found to be specifically enriched for the sequences previously identified as prophage regions. Heat stress (50°C, 30 min) was also found to induce phage release in V. campbellii. Notably, promoter activity of two representative phage genes indicated heterogeneous phage induction within the population.