Visualizing the molecular sociology at the HeLa cell nuclear periphery

Mahamid, Julia; Pfeffer, Stefan; Schaffer, Miroslava; Villa, Elizabeth; Danev, Radostin; Cuellar, Luis Kuhn; Förster, Friedrich; Hyman, Anthony A.; Plitzko, Jürgen M.; Baumeister, Wolfgang

doi:10.1126/science.aad8857

Cited by 540 publications

(485 citation statements)

References 43 publications

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“…Electron microscopy clearly shows that microtubules closely associate with NPCs (Gray and Westrum, 1976;Mahamid et al, 2016;Fig. 2) and there is increasing evidence that this is an actual physical linkage via motor proteins, with physiological, variable, complex and not fully understood functions.…”

Section: Microtubules Are Physically Linked To the Npcmentioning

confidence: 99%

“…Thin section electron microscopy (Gray and Westrum, 1976) and cryo electron tomography (Mahamid et al, 2016) have shown that the cytoskeleton is in close association with the NPCs. Deep etch electron microscopy has also indicated an association of intermediate filaments, possibly vimentin, with the cytoplasmic ring of the NPC (Fujitani et al, 1988).…”

Section: Actin and Cytoplasmic Intermediate Filaments Associate With mentioning

confidence: 99%

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Nuclear pore complex tethers to the cytoskeleton

Goldberg

2017

Seminars in Cell & Developmental Biology

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Section: Microtubules Are Physically Linked To the Npcmentioning

confidence: 99%

Section: Actin and Cytoplasmic Intermediate Filaments Associate With mentioning

confidence: 99%

Nuclear pore complex tethers to the cytoskeleton

Goldberg

2017

Seminars in Cell & Developmental Biology

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“…Hence, it is highly desirable to study them in situ -within an unperturbed cell in which they perform their function. Recent advances in cryo-focused ion beam (FIB) micromachining bring 'large' mammalian cells into the range of electron transparency (100-300 nm) [1], and cryo-electron tomography (ET) on these preparations now provides marvelous molecular views in cells [2].Interpretation of these views within the context of specific cellular functions is the next goal [3]; the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Thus, retaining low-abundance and dynamic subcellular structures, arrested by vitrification, within such limited volumes requires precise targeting of the FIB milling process.…”

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Site Specific Cryo-FIB Preparations Aimed at in situ Cryo-Electron Tomography

2017

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The function of many macromolecular assemblies is so deeply rooted in their cellular context that it is impossible to isolate them without compromising their structural integrity. Hence, it is highly desirable to study them in situ -within an unperturbed cell in which they perform their function. Recent advances in cryo-focused ion beam (FIB) micromachining bring 'large' mammalian cells into the range of electron transparency (100-300 nm) [1], and cryo-electron tomography (ET) on these preparations now provides marvelous molecular views in cells [2].Interpretation of these views within the context of specific cellular functions is the next goal [3]; the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Thus, retaining low-abundance and dynamic subcellular structures, arrested by vitrification, within such limited volumes requires precise targeting of the FIB milling process. To answer these needs, we describe the incorporation of confocal light microscopy (LM) at cryogenic temperatures into existing workflows for cellular sample preparation by cryo-FIB (Figure 1). By introducing microspheres as fiducial markers that are distinguished both in LM and the scanning electron microscope (SEM)/FIB, we compute a 3D 'rigid-body' coordinate transformation. The correlation accuracy attained with the aid of fiducial markers surpasses the physical resolution of the LM and allows targeting within the range of tens of nanometers.A software encompassing the necessary functions to calculate 3D correlation is under open source development ([4] and Figure 1: II). This includes data preprocessing tools of LM confocal stacks and 3D coordinate extraction functions that determine the location of markers in the LM volume semiautomatically. Appropriate sample positions, judged by the number and distribution of markers, are selected prior to the SEM/FIB session. Markers in the SEM and FIB images are then selected manually by the user, which introduces errors and a possible propagation of uncertainty. Thus, after an initial correlation, visual guides indicate deviation of the selected markers from their respective calculated positions. This helps the user to identify manual selection bias, eliminate markers with large deviations and allows for adjustments, leading to minimization of those errors. The process is conducted while operating the SEM/FIB and allows for on-line 3D correlation and immediate site-specific cryo-FIB preparation (Figure 1: III).The correlative approach was employed to navigate FIB milling and to capture specific structures, lipid droplets as an example, in vitrified cellular samples [3]. The correlation approach can then be applied to navigate the acquisition of cryo-ET data within the FIB-lamella at specific locations, unambiguously identified by fluorescence microscopy (Figure 1: IV).We will discuss approaches to improve the localization specificity and precision in 3D, the preparation reproducibility and yield, all together key factors in advancing in situ im...

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New Scios Cryo^TM - Dedicated FIB/SEM for Advanced Cryo-Lamella Preparation in Structural Biology

2017

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We would like to introduce the new FEI Scios Cryo; a system which is built upon a successful prototype and other variants which have resulted already in significant publications [1,2,3]. This instrument offers customers a dedicated and reliable entry point to the world of 'in situ' lamella preparation for cryo electron tomography (cryoET). Cryo-focused ion beam (cryo-FIB)-based sample preparation has recently been shown to extend structural biology from solely purified structures, to the dynamic complexity of the cell [4].Cells typically need to be thinned to a thickness which is conducive for modern cryo-TEMs. To this end researchers have recently been exploring the use of DualBeam TM microscopes to enable thinning of frozen-hydrated biological cells. Such FIB-SEM instruments utilize a primary electron column for imaging and a secondary ion column for milling. Typically Gallium ions are focused onto the sample in order to facilitate removal of material [5].The Scios Cryo consists of the following modules: A Scios 2 DualBeam TM platform, dedicated and equipped with a cryo loading system, as well as an innovative cryo stage with full rotation and tilt capabilities. Furthermore, a cryo cooling system involving a heat exchanger for cooling of nitrogen gas, a preparation station for loading and unloading samples under cryogenic conditions and finally, software which has dedicated functionality to assist the users in producing better quality lamellae. Figure 1 shows a schematic of the modules.Plunge frozen cells which have been transferred to an FEI Autogrid can be loaded on to a shuttle under cryogenic conditions and transferred in the DualBeam TM . Here the sample can be correlated to optical data using Maps TM software, allowing for identification of specific cells which have the desired phenotype. Cells can be identified and regions of interest can be selected. From here the user can navigate to the desired cell by merely clicking in an overview image.Sputtered inorganic metal layers are used to facilitate conduction, which can be done inside the main chamber. Protective layers of organometallic platinum can be added to allow milling of straight edges without damage from stray ions. Typically a milling position, which is at a slight elevation with respect to the TEM grid, is chosen resulting in an in situ lamella which is as close to the plane with the grid as possible. Examples are shown in figure 2. These lamellae can be transferred out of the microscope into the loading station, where the grid can be transferred to the Talos Artica TM or Titan Krios TM TEM's allowing collection of tomographic data. The Scios Cryo complements our integrated product suite, providing a complete cryo-tomography workflow, by adding a dedicated preparation tool for vitreous sample thinning for structural biology.

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Visualizing the molecular sociology at the HeLa cell nuclear periphery

Cited by 540 publications

References 43 publications

Nuclear pore complex tethers to the cytoskeleton

Nuclear pore complex tethers to the cytoskeleton

Site Specific Cryo-FIB Preparations Aimed at in situ Cryo-Electron Tomography

New Scios Cryo^TM - Dedicated FIB/SEM for Advanced Cryo-Lamella Preparation in Structural Biology

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Visualizing the molecular sociology at the HeLa cell nuclear periphery

Cited by 540 publications

References 43 publications

Nuclear pore complex tethers to the cytoskeleton

Nuclear pore complex tethers to the cytoskeleton

Site Specific Cryo-FIB Preparations Aimed at in situ Cryo-Electron Tomography

New Scios CryoTM - Dedicated FIB/SEM for Advanced Cryo-Lamella Preparation in Structural Biology

Contact Info

Product

Resources

About

New Scios Cryo^TM - Dedicated FIB/SEM for Advanced Cryo-Lamella Preparation in Structural Biology