Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging

Sousa, Alioscka A.; Azari, Afrouz A; Zhang, Guofeng; Leapman, Richard D.

doi:10.1016/j.jsb.2010.10.017

Cited by 73 publications

(42 citation statements)

References 27 publications

(34 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Reconstructions of the entire PSD by STEM tomography on thick sections (62,63) helps to evaluate the global effects of the knockdown on the sizes of PSDs and synaptic terminals. Data from electrophysiology and thin-and thicksection EM tomography illustrate how MAGUKs play essential roles in maintaining the overall morphology of the PSD and in anchoring glutamate receptors at the PSD (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, on average, only one-third or less of the PSD is sampled, which might lead to biases in the 2D EM data. These limitations can be overcome by making 3D reconstructions of sections 1-to 2-μm-thick with scanning transmission EM (STEM) tomography, which avoids the chromatic aberration that blurs images in thick sections (62,63). The thicker sections contain entire PSDs, allowing the PSDs to be completely reconstructed without serial sectioning (Fig.…”

Section: Minimal Structural Changes In Pre-and Postsynaptic Membranesmentioning

confidence: 99%

See 1 more Smart Citation

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Chen

Levy

Hou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

238

201

View full text Add to dashboard Cite

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre-or postsynaptic membrane, and depletes the number of membraneassociated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membraneassociated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.T he postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2-5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6-10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14-18). These MAGUK proteins share conserved modular structures consisting of three...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Minimal Structural Changes In Pre-and Postsynaptic Membranesmentioning

confidence: 99%

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Chen

Levy

Hou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

238

201

View full text Add to dashboard Cite

show abstract

“…These consist of variations on either tilt-series electron tomography or serial-block-face scanning electron microscopy. In tilt series electron tomography, either standard beam geometry is used or STEM (scanning transmission electron microscopy) geometry [24–25]. In standard geometry, section thickness is limited to about 300 nm because electron scattering increases with section thickness.…”

Section: New Standards For Imaging Plateletsmentioning

confidence: 99%

The cellular basis of platelet secretion: Emerging structure/function relationships

Yadav

Storrie

2016

Platelets

View full text Add to dashboard Cite

Platelet activation has long been known to be accompanied by secretion from at least three types of compartments. These include dense granules, the major source of small molecules; α-granules, the major protein storage organelle, and lysosomes, the site of acid hydrolase storage. Despite ~60 years of research, there are still many unanswered questions about the cell biology of platelet secretion: e.g., how are these secretory organelles organized to support cargo release and what are the key routes of cargo release, granule to plasma membrane or granule to canalicular system (CS). Moreover, in recent years, increasing evidence points to the platelet being organized for secretion of the contents from other organelles, namely, the dense tubular system (endoplasmic reticulum) and the Golgi apparatus. Conceivably, protein secretion is a wide-spread property of the platelet and its organelles. In this review, we concentrate on the cell biology of the α-granule and its structure/function relationships. We both review the literature and discuss the wide array of 3-dimensional, high resolution structural approaches that have emerged in the last few years. These have begun to reveal new and unanticipated outcomes and some of these are discussed. We are hopeful that the next several years will bring rapid advances to this field that will resolve past controversies and be clinically relevant.

show abstract

“…Furthermore, the scanning electron probe may be focused anywhere on the sample, even at high tilt angles, which will generally have areas out of focus with conventional TEM imaging [17]. Also available in STEM mode, axial BF imaging, as demonstrated by Sousa et al [18] in the investigation of pancreatic beta cells, has the ability to extend the thickness of investigated biological specimens up to 1 mm. Similar outcomes were noted by Hohmann-Marriott et al [17] when they used axial BF-STEM tomography to study eukaryotic cells.…”

Section: Tomographic Techniquesmentioning

confidence: 99%

High-resolution three-dimensional probes of biomaterials and their interfaces

Grandfield

Palmquist

Engqvist

2012

Phil. Trans. R. Soc. A.

View full text Add to dashboard Cite

Interfacial relationships between biomaterials and tissues strongly influence the success of implant materials and their long-term functionality. Owing to the inhomogeneity of biological tissues at an interface, in particular bone tissue, two-dimensional images often lack detail on the interfacial morphological complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar length scale. Electron tomography (ET) can meet these challenges by enabling high-resolution three-dimensional imaging of biomaterial interfaces. In this article, we review the fundamentals of ET and highlight its recent applications in probing the three-dimensional structure of bioceramics and their interfaces, with particular focus on the hydroxyapatite-bone interface, titanium dioxide-bone interface and a mesoporous titania coating for controlled drug release.

show abstract

Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging

Cited by 73 publications

References 27 publications

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

The cellular basis of platelet secretion: Emerging structure/function relationships

High-resolution three-dimensional probes of biomaterials and their interfaces

Contact Info

Product

Resources

About