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16 Janelia Publications
Showing 1-10 of 16 resultsVimentin intermediate filaments (VIFs) form complex, tightly packed networks; due to this density, traditional imaging approaches cannot discern single-filament behavior. To address this, we developed and validated a sparse vimentin-SunTag labeling strategy, enabling single-particle tracking of individual VIFs and providing a sensitive, unbiased, and quantitative method for measuring global VIF motility. Using this approach, we define the steady-state VIF motility rate, showing a constant ∼8% of VIFs undergo directed microtubule-based motion irrespective of subcellular location or local filament density. Significantly, our single-particle tracking approach revealed uncorrelated motion of individual VIFs within bundles, an observation seemingly at odds with conventional models of tightly cross-linked bundles. To address this, we acquired high-resolution focused ion beam scanning electron microscopy volumes of vitreously frozen cells and reconstructed three-dimensional VIF bundles, finding that they form only loosely organized, semi-coherent structures from which single VIFs frequently emerge to locally engage neighboring microtubules. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time. bioRxiv Preprint: https://doi.org/10.1101/2024.06.10.598346
Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at ∼1 μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca channels and ER Ca-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca homeostasis, and local activation of the Ca/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca release at ER-PM junctions over 20 μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.
Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.
Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.
To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.
To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites3. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle4,5. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation6,7, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.
Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.
Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM). We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, 'OpenOrganelle', to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.
Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.