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MamK, an actin-like protein conserved in magnetotactic bacteria, functions as a cytoskeletal element that positions the magnetosome, a bacterial geomagnetic sensor organelle. Specifically, MamK polymerizes into filaments associated with the magnetosome chain within each cell. The dynamics of these filaments are fundamental to magnetosome organelle positioning. However, the dynamic nature of the polymerized MamK filaments has not been characterized at the molecular level under physiological conditions. In this study, we used high-speed atomic force microscopy (AFM) to characterize the dynamic MamK polymerization process. MamK polymerized as double-helical filaments with a half-helical pitch distance of 41.3 ± 5.1 nm and a filament diameter of 6.3 ± 0.5 nm. The polymerizing MamK filaments elongated at average speeds of 12.4 ± 4.2 nm/min at the fast-growth ends (plus ends) and from − 4.4 to 8.0 nm/min at the slow-growth ends (minus ends) on the mica substrate in the solution containing 3 µM monomeric MamK. High-speed AFM demonstrated that MamK polymerized into dynamic double-helical filaments with polarity similar to that of eukaryotic actin filaments. Understanding the intrinsic dynamics of MamK, a well-conserved actin-like protein in magnetotactic bacteria, is key to elucidating the mechanism of magnetosome positioning in a bacterial cell.

Magnetotactic bacteria are a diverse group of microorganisms that use intracellular chains of ferrimagnetic nanocrystals, produced within magnetosome organelles, to align and navigate along the geomagnetic field. Several conserved genes for magnetosome formation have been described, but the mechanisms leading to distinct species-specific magnetosome chain configurations remain unclear. Here, we show that the fragmented nature of magnetosome chains in Magnetospirillum magneticum AMB-1 is controlled by genes mcaA and mcaB . McaA recognizes the positive curvature of the inner cell membrane, while McaB localizes to magnetosomes. Along with the MamK actin-like cytoskeleton, McaA and McaB create space for addition of new magnetosomes in between pre-existing magnetosomes. Phylogenetic analyses suggest that McaA and McaB homologs are widespread among magnetotactic bacteria and may represent an ancient strategy for magnetosome positioning. Magnetotactic bacteria use intracellular chains of ferrimagnetic nanocrystals, produced within magnetosome organelles, to align and navigate along the geomagnetic field. Here, Wan et al. identify two proteins involved in magnetosome positioning in Magnetospirillum magneticum , homologs of which are widespread among magnetotactic bacteria.

Small extracellular vesicles (sEVs), which carry lipids, proteins and RNAs from their parent cells, serve as biomarkers for specific cell types and biological states. These vesicles, including exosomes and microvesicles, facilitate intercellular communication by transferring cellular components between cells. Current methods, such as ultracentrifugation and Tim‐4 affinity method, yield high‐purity sEVs. However, despite their small size, purified sEVs remain heterogeneous due to their varied intracellular origins. In this technical note, we used high‐speed atomic force microscopy (HS‐AFM) in conjunction with exosome markers (IgGCD63 and IgGCD81) to explore the intracellular origins of sEVs at single‐sEV resolution. Our results first revealed the nanotopology of HEK293T‐derived sEVs under physiological conditions. Larger sEVs (diameter > 100 nm) exhibited greater height fluctuations compared to smaller sEVs (diameter ≤ 100 nm). Next, we found that mouse‐origin IgGCD63, and rabbit‐origin IgGcontrol and IgGCD81, exhibited the iconic ‘Y’ conformation, and similar structural dynamics properties. Last, exosome marker antibodies predominantly co‐localised with sEVd ≤ 100 nm but not with sEVd > 100 nm, demonstrating the CD63‐CD81‐enriched sEV and CD63‐CD81‐depleted sEV subpopulations. In summary, we demonstrate that nanoscopic profiling of surface exosome markers on sEVs using HS‐AFM is feasible for characterising distinct sEV subpopulations in a heterogeneous sEV mixture.

Magnetotactic bacteria are a unique group of bacteria that synthesize a magnetic organelle termed the magnetosome, which they use to assist with their magnetic navigation in a specific type of bacterial motility called magneto-aerotaxis. Cytoskeletal filaments consisting of the actin-like protein MamK are associated with the magnetosome chain. Previously, the function of MamK was thought to be in positioning magnetosome organelles; this was proposed based on observations via electron microscopy still images. Here, we conducted live-cell time-lapse fluorescence imaging analyses employing highly inclined and laminated optical sheet microscopy, and these methods enabled us to visualize detailed dynamic movement of magnetosomes in growing cells during the entire cell cycle with high-temporal resolution and a high signal/noise ratio. We found that the MamK cytoskeleton anchors magnetosomes through a mechanism that requires MamK-ATPase activity throughout the cell cycle to prevent simple diffusion of magnetosomes within the cell. We concluded that the static chain-like arrangement of the magnetosomes is required to precisely and consistently segregate the magnetosomes to daughter cells. Thus, the daughter cells inherit a functional magnetic sensor that mediates magneto-reception. IMPORTANCE Half a century ago, bacterial cells were considered a simple “bag of enzymes”; only recently have they been shown to comprise ordered complexes of macromolecular structures, such as bacterial organelles and cytoskeletons, similar to their eukaryotic counterparts. In eukaryotic cells, the positioning of organelles is regulated by cytoskeletal elements. However, the role of cytoskeletal elements in the positioning of bacterial organelles, such as magnetosomes, remains unclear. Magnetosomes are associated with cytoskeletal filaments that consist of the actin-like protein MamK. In this study, we focused on how the MamK cytoskeleton regulates the dynamic movement of magnetosome organelles in living magnetotactic bacterial cells. Here, we used fluorescence imaging to visualize the dynamics of magnetosomes throughout the cell cycle in living magnetotactic bacterial cells to understand how they use the actin-like cytoskeleton to maintain and to make functional their nano-sized magnetic organelles. Half a century ago, bacterial cells were considered a simple “bag of enzymes”; only recently have they been shown to comprise ordered complexes of macromolecular structures, such as bacterial organelles and cytoskeletons, similar to their eukaryotic counterparts. In eukaryotic cells, the positioning of organelles is regulated by cytoskeletal elements. However, the role of cytoskeletal elements in the positioning of bacterial organelles, such as magnetosomes, remains unclear. Magnetosomes are associated with cytoskeletal filaments that consist of the actin-like protein MamK. In this study, we focused on how the MamK cytoskeleton regulates the dynamic movement of magnetosome organelles in living magnetotactic bacterial cells. Here, we used fluorescence imaging to visualize the dynamics of magnetosomes throughout the cell cycle in living magnetotactic bacterial cells to understand how they use the actin-like cytoskeleton to maintain and to make functional their nano-sized magnetic organelles.

Magnetotactic bacteria are ubiquitous aquatic prokaryotes that have the ability to biomineralize magnetite (Fe 3 O 4 ) and/or greigite (Fe 3 S 4 ) nanoparticles called magnetosomes. Magnetotactic cocci belonging to the class “ Ca . Magnetococcia” are most frequently identified in freshwater habitats, but remain uncultivated. Here, we report for the first time axenic cultivation of freshwater magnetotactic coccus FCR-1 isolated from Chichijima, Japan. Strain FCR-1 grows microaerophilically in a semi-solid gellan gum medium. We find that strain FCR-1 biomineralizes Fe 3 O 4 nanoparticles, which are not chained, into a cell. Based on phylogenomic analysis, compared with strains of the class “ Ca . Magnetococcia”, strain FCR-1 represents a novel genus of candidate family “ Ca . Magnetaquicoccaceae” within the class “ Ca . Magnetococcia” and we tentatively name this novel genus “ Ca . Magnetaquiglobus chichijimensis”. Our isolate provides a promising tool for elucidating the functions of unchained magnetosomes, the global distribution of magnetotactic bacteria and the origin of magnetotaxis. Magnetotactic coccus FCR-1, axenically cultivated, is a novel freshwater magnetotactic coccus belonging to the family Ca. Magnetaquicoccaceae and biomineralizes unchained truncated hexagonal prismatic Fe3O4 nanoparticles.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

The unique ability of magnetotactic bacteria to navigate along a geomagnetic field is accomplished with the help of prokaryotic organelles, magnetosomes. The magnetosomes have well-ordered chain-like structures, comprising membrane-enveloped, nanosized magnetic crystals, and various types of specifically associated proteins. In this study, we applied atomic force microscopy (AFM) to investigate the spatial configuration of isolated magnetosomes from Magnetospirillum magneticum AMB-1 in near-native buffer conditions. AFM observation revealed organic material with a ∼7-nm thickness surrounding a magnetite crystal. Small globular proteins, identified as magnetosome-associated protein MamA, were distributed on the mica surface around the magnetosome. Immuno-labeling with AFM showed that MamA is located on the magnetosome surface. In vitro experiments showed that MamA proteins interact with each other and form a high molecular mass complex. These findings suggest that magnetosomes are covered with MamA oligomers in near-native environments. Furthermore, nanodissection revealed that magnetosomes are built with heterogeneous structures that comprise the organic layer. This study provides important clues to the supramolecular architecture of the bacterial organelle, the magnetosome, and insight into the function of the proteins localized in the organelle.

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P 50 ) and 1.96 ( n value), and a small Bohr effect (δH +  = −0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O 2 affinity at P 50 of 2.5 mmHg which may assure efficient utilization of the lung O 2 reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl 2 ·6H 2 O] solution at a pH of 7.0–8.5.

Magnetosomes serve as a model for understanding the cell biology of bacterial organelles and the mechanisms of bacterial cell organization. MamK, one of the best-studied bacterial actins, is a notable player in magnetosome chain assembly. This work explores how MamK dynamics are altered by two potential bacterial actin-binding proteins, McaA and McaB. This system illustrates how changes in bacterial cytoskeleton regulation result in different organization of subcellular compartments. The conclusions from this research also have implications for understanding the broader evolutionary strategies for the regulation of actin-like proteins and diversification of compartment organization in bacteria.